Methods and compositions for inducing apoptosis by stimulating er stress

ABSTRACT

The present invention provides a method for inducing apoptosis in selected cells by aggravating ER-stress. The aggravation of ER-stress is achieved in a specific manner by inhibiting SERCA (sarcoplasmic/endoplasmic reticulum calcium ATPase), leading to elevated level of cytoplasmic calcium concentration, yet without inhibiting the activity of COX-2 (cyclooxygenase-2) or triggering the release of histamine. Induction of apoptosis may be enhanced by first inducing or further aggravating ER-stress through inhibition of proteasome or proteases. Also provided are compounds and compositions useful as ER-stress aggravating agents, methods for screening, selecting, identifying and designing the same and methods for treating diseased conditions by inducing apoptosis through specific and selective aggravation of ER-stress.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims an invention which was disclosed in ProvisionalApplication No. 60/908,107, filed Mar. 26, 2007, entitled “USE OFCHEMICAL COMPOUNDS TO STIMULATE THE ENDOPLASMIC RETICULUM (ER) STRESSRESPONSE IN TUMOR CELLS CAUSING TUMOR CELL DEATH”. The benefit under 35USC §119(e) of the United States provisional application is herebyclaimed. The above priority application is hereby incorporated herein byreference.

FIELD OF THE INVENTION

The invention pertains to the field of molecular medicine. Moreparticularly, the invention pertains to methods, compounds, andcompositions for selectively inducing apoptosis in cells by aggravatingthe ER stress response. The invention also relates to methods for thetreatment of cancer, including difficult to treat, drug-resistant, andrecurring cancer.

BACKGROUND OF THE INVENTION

Apoptosis is a process involving programmed cell death or “cellularsuicide” and under physiological conditions it occurs after cell surfacedeath receptors are occupied or activated by various genotoxic agents.This process leads to mitochondrial release of cytochrome C, which inturn activates the caspase enzymes that promote apoptosis.

Since apoptosis was first described in 1972 by Kerr et al. (1), muchknowledge has been accumulated about this important cellular process.Although a comprehensive understanding of apoptosis at the molecularlevel and cellular level is yet to be achieved, the knowledgeaccumulated thus far has led to the realization that because the processis genetically programmed, it may be susceptible to the effects ofmutation and, therefore, may be involved in the pathogenesis of avariety of human diseases such as viral infections, autoimmune diseasesand cancer (2, 3). Based on this realization, it has been widelyrecognized that any therapeutic strategy aimed at specificallytriggering apoptosis in diseased cells that suffer from disregulation ofapoptosis (e.g. cancer) may deliver potentially promising therapies.

In a recent article, Ferreira et al. (4) have reviewed currentstrategies for exploiting the therapeutic potentials of apoptosis. Tosummarize the review, existing strategies for apoptosis-based therapiescan be grouped into two types: the proapoptotic approaches and theapoptosis-permissive approaches. Table 1 shows a list of exemplaryapproaches in each category.

The proapoptic approaches are strategies that aim to directly induceapoptosis. They try to achieve apoptosis through exploitation ofexisting cellular players and pathways such as death receptors andcaspases, or the introduction of exogenous proapoptotic molecules suchas Apoptin. Proapoptotic strategies can involve: (a) direct introductionof proapoptotic players; (b) modulation of antiapoptotic molecules; or(c) restoration of tumor suppressor gene function. However, proapoticstrategies are not based on structural differences between normal andcancer cells. Therefore, achieving tumor cell specificity, whileminimizing toxicity, poses a major challenge in the development of thistype of approaches For example, experimental therapies targeting deathreceptors such as TNF and Fas have resulted in ischemic and hemorrhagiclesions in several tissues.

The apoptosis-permissive approaches, on the other hand, is based on thepremises that by blocking some of the intricate signaling pathwaysmediating survival messages that in normal conditions contribute to keepthe cellular homeostasis, apoptosis may be triggered. This type ofstrategies do not have the non-specific toxicity problems of theproapoptotic approaches, however, successful development of this type ofstrategies is highly dependent on a detailed knowledge of the mechanismsby which apoptosis is facilitated. Thus far, our understanding andknowledge of such mechanisms leading to the secondary effect ofapoptosis are still incomplete.

Therefore, there still exists a need for new strategies to selectivelyinduce apoptosis in diseased cells, and new tools and methods forimplementing the same.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide a methodfor inducing apoptosis that may serve as the basis for new therapies.

A further object of the present invention is to provide compounds andcompositions that are useful as apoptosis inducing agents.

Another further object of the present invention is to provide a methodfor screening, selecting, and discovering compounds that are useful asagents for inducing apoptosis.

Yet another further object of the present invention is to provideapoptosis-based treatment methods for treating diseases.

These and other objects of the present invention, which will become moreapparent in conjunction with the following detailed description of thepreferred embodiments, either along or in combinations thereof, havebeen satisfied by the unexpected discovery of a new endoplasmicreticulum (ER)-stress response mechanism which leads to apoptosis.

In a first aspect, the present invention provides a method of inducingor aggravating stress in a cell's ER to trigger apoptosis. Embodimentsaccording to this aspect of the present invention generally include thesteps of selectively inhibiting sarcoplasmic/ER calcium ATP-ase (SERCA)activity in the cell without inhibiting cyclooxygenase-2 (COX-2)activity; and elevating the expression of CCAAT/enhancer binding proteinhomologous transcription factor, also called GADD153: growth arrest andDNA damage-inducible gene 153 (CHOP) in the cell. The combination ofinhibiting SERCA activity and elevating CHOP expression results in acondition favorable for initiation of apoptosis in the cell.

In a second aspect, the present invention provides a method forscreening, selecting, or designing compounds useful for inducing oraggravating ER stress in a cell to trigger apoptosis in the cell.Embodiments according to this aspect of the present invention generallyinclude the steps of obtaining information about a test compound andidentifying the test compound as a potential ER-stress aggravating agentif the compound is an inhibitor of SERCA, and is not an inhibitor ofCOX-2.

In a third aspect, the present invention provides compounds useful as anER stress aggravating agent, having the general formula:

wherein,

-   R₁ is methyl, fluoromethyl, difluoromethyl, trifluoromethyl, alkyl,    fluoroalkyl, difluoroalkyl, trifluoroalkyl, polyfluoroalkyl,    hydroxyalkyl, or carboxyalkyl;-   R₂ is hydrogen, fluoro, chloro, bromo; fluoromethyl, difluoromethyl,    trifluoromethyl, alkyl, fluoroalkyl, difluoroalkyl, trifluoroalkyl,    polyfluoroalkyl, hydroxyalkyl, or carboxyalkyl;-   R₃-R₇ are independently selected from a group consisting of:    hydrogen, fluoro, chloro, bromo, alloxy, alkyl, fluoroalkyl,    difluoroalkyl, trifluoroalkyl, polyfluoroalkyl, hydroxyalkyl, or    carboxyalkyl, aryl and heteroaryl;-   R₈-R₁₁ are independently selected from a group consisting of:    hydrogen, fluoro, chloro, bromo; fluoromethyl, difluoromethyl,    trifluoromethyl, alkyl fluoroalkyl, difluoroalkyl, trifluoroalkyl,    polyfluoroalkyl, hydroxyalkyl, carboxyalkyl, alkenyl, alkynyl,    cycloalkyl, heterocyclic, aryl, or heteroaryl; and-   R₁₂ is hydrogen, acetyl, acyl, alkyl, fluoroalkyl, difluoroalkyl,    trifluoroalkyl, polyfluoroalkyl, hydroxyalkyl, carboxyalkyl;    aminoacyl, aminoalkyl, cycloalkyl, heterocyclic, aryl, or    heteroaryl.

In a fourth aspect, the present invention also provides a pharmaceuticalcomposition useful for inducing or aggravating ER stress in a cell totrigger apoptosis in the cell. Embodiments according to this aspect ofthe present invention generally include an ER-stress aggravating agent;and a pharmaceutically acceptable carrier.

In a fifth aspect, the present invention also provides a method fortreating a diseased condition in a patient by inducing or aggravating ERstress in selected diseased cells to trigger apoptosis in the cells.Embodiments according to this aspect of the present invention generallyinclude the steps of: administering a pharmaceutically effective amountof a pharmaceutical composition according to the previous aspect of thepresent invention.

Other aspects and advantages of the invention will be apparent from thefollowing description and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a simplified model of the critical events during the ERstress response. In case of severe ER stress (such as after theinhibition of SERCA) translational attenuation ensues and participatesin the subsequent events of the ER stress response (ESR). The ESR isconstituted of two antagonistic parts: (1) protective components (listedin the left box) that execute defensive processes to ensure cellularsurvival under stress, and (2) pro-apoptosis (death-inducing) components(listed in the right box), which will begin to dominate and trigger celldeath (apoptosis) if stress becomes too excessive or cannot be relieved(such as in the continuous presence of a SERCA inhibitor). In case oflow-level/chronic ER stress, as is frequently present in tumor cells,components in the left box maintain dominance and support tumor cellsurvival even under adverse conditions that are oftentimes present intumor tissue (low levels of oxygen, low glucose levels); under theseconditions, the components shown in the right box are either not presentor are only weakly active. However, when the tumor cell encountersadditional ER stress, such as through the pharmacologic inhibition ofSERCA, the already present low-level ER stress will be greatlyaggravated and the components shown in the right box become activatedand gain dominance; under these conditions, the defensive effort ofcomponents in the left box is being overwhelmed, and the components inthe right box will execute tumor cell death. Note that the listedcaspase 12 refers to the murine form of this enzyme; the humanorthologue is caspase 4 (i.e., human caspase 4 and mouse caspase 12execute similar functions during the events of the ESR).

FIG. 2 illustrates the three major activity levels of the ER stressresponse system and the antagonistic defensive/pro-apoptotic functionsof GRP78 and CHOP: (A) The “No ER Stress” condition (left) is thedefault situation in normal cells. Here, very low levels of GRP78 in theER lumen bind to PERK, IRE1, and ATF6, and keep these ER stresscomponents in their inactive states. (B) In cancer cells, chronic stress(low glucose, hypoxia, misfolded proteins) generates the “Low ER Stress”condition (middle). Here, low levels of continuous stress lead to thepartial activation of the ER stress response system, with emphasis onelevated levels of GRP78 (thickened arrows), which provide theprotective component of the ER stress system and furthermore increasechemo-resistance of tumor cells. In this scenario, the ER transmembranecomponents PERK, IRE1, and ATF6 display low levels of activity, whichpresumable are fine-tuned and adjusted by GRP78. Elevated levels ofGRP78 help to neutralize deleterious effects of the initial stresscondition, such as through the ability to act as a chaperone formisfolded proteins. (C) Persistent, high level stress generates the“Severe ER Stress” situation (right), which is characterized by severe(but transient) inhibition of protein synthesis via PERK-mediatedphosphorylation (=inactivation) of translation initiation factor 2 alpha(eIF2α) (thick lines). Under these conditions, the protective effort ofGRP78 is overwhelmed, and the activation of pro-apoptotic CHOP andsubsequent initiation of cell death dominates (very thick arrows). Ourwork has demonstrated that during treatment of tumor cells with DMC, the“Severe ER Stress” scenario applies. In the continuous presence of thisdrug, the cell is unable to neutralize drug-induced stress, despiteelevated levels of GRP78; instead, strong induction of CHOP andactivation of caspase 4 initiate apoptosis, and the cell dies.

FIG. 3 shows that Celecoxib and DMC induce CHOP protein levels invarious cancer cell lines. Several different cancer cell lines (asindicated on the left) were cultured in the presence of celecoxib (Cxb;40 and 60 μM) or DMC (30 and 50 μM) for 48 hours (Co: control,non-treated cells). Total cellular lysates were prepared and analyzed byWestern blot analysis with specific antibodies to CHOP. As a control forequal loading, all blots were also analyzed with antibodies to actin(only one of these control blots is shown at the bottom). The tumor typeof each cell line is indicated on the right.

FIG. 4 shows that ER stress indicators are induced similarly by DMC andthapsigargin. U251 glioblastoma cells were cultured in the presence of 1μM thapsigargin (Tg) or 60 μM DMC for various times, as indicated. Totalcell lysates were prepared and analyzed by Western blot with specificantibodies to the ER stress proteins GRP78, CHOP, and caspase 4 (Casp4). Actin was used as a loading control. Pro-casp 4 denotes the inactivecaspase 4 proenzyme, whereas Cleaved Casp 4 is indicative of theactivated form of this enzyme; * refers to a faster-migrating band thatis inconsistently observed in these Western blots.

FIG. 5 shows that DMC and celecoxib, but not other coxibs or NSAIDs,induce calcium release into the cytoplasm. (A) U251 cells were treatedwith DMC or various coxibs and NSAIDs, and changes in intracellularcalcium levels were recorded as described in Materials & Methods. Thetop two panels show the typical spikes of calcium increase that wereconsistently observed in response to DMC or celecoxib treatment. Thethird panel shows the typical response (i.e., lack thereof) tovaldecoxib, rofecoxib, flurbiprofen, indomethacin, and sulindac (onlyshown for valdecoxib). Arrows indicate timepoint of drug addition. (B)Chart shows the average (mean±SD) calcium increase in response totreatment with the various drugs from several repeats. Essentiallysimilar results were also obtained with the LN229 cell line.

FIG. 6 shows that the induction of CHOP and GRP78 is specific tocelecoxib and DMC. U251 glioblastoma cells were cultured in the presenceof DMC, celecoxib, rofecoxib, or valdecoxib, and the protein levels ofCHOP and GRP78 were determined by Western blot analysis. Shown are (A)time kinetics at 50 μM of each drug, and (B) concentration dependenceafter 15 hours of incubation. Bgr. refers to a background signal that isinconsistently observed with the GRP78 antibody. All blots wereprocessed in parallel, so that signal intensity is directly comparableamong the different panels. Note that DMC is the most potent stimulant,whereas rofecoxib and valdecoxib are inactive under these conditions.

FIG. 7 shows that the induction of CHOP and GRP78 is specific tocelecoxib and DMC and requires calcium. U251 glioblastoma cells werecultured in the presence of DMC, celecoxib, rofecoxib, or valdecoxib,and the protein levels of CHOP and GRP78 were determined by Western blotanalysis. Shown are (A) time kinetics at 50 μM of each drug, and (B)concentration dependence after 15 hours of incubation. Bgr. refers to abackground signal that is inconsistently observed with the GRP78antibody. In (C), cells were treated with 60 μM DMC in the presence orabsence of 20 μM BAPTA-AM and 0.78 mM EGTA, both of which are potentchelators of Ca²⁺. All blots in A and B were processed in parallel, sothat signal intensity is directly comparable among the different panels.

FIG. 8 shows that the induction of CHOP and GRP78 correlates withincreased apoptosis and reduced cell growth and survival. U251glioblastoma cells were treated with different drugs, and variousparameters of cell growth and cell death were comparatively analyzed. Ascontrols, cells remained either non-treated (Co) or were treated withthe solvent DMSO alone. (A) Cells were treated with 30 or 50 μM DMC for48 hours and the effects on cell growth/survival and on cell death weredetermined by various assays. The panel labeled Number of Coloniesdisplays the results from a colony forming assay, where the absolutenumber of surviving cells able to spawn a colony of newly grown cellswas determined. The panel labeled % Cell Growth and Survival shows theresults of conventional MTT assays performed at the end of the 48-hourdrug treatment period. The panel labeled % Apoptotic Cells presents thepercentage of cells undergoing apoptosis as revealed by the TUNEL assayafter 48 hours of drug treatment. Below the three panels, the expressionlevels of the ER stress indicators CHOP, GRP78, and caspase 4 at the endof the 48 hour drug treatment is shown, as determined by Western blotanalysis with specific antibodies (actin served as a loading control.(B) Cells were treated with various concentrations of different drugsfor 48 hours, as indicated, and cell death was measured with the celldeath ELISA kit. (C) Cells were treated with DMC or various coxibs andtraditional NSAIDs for 48 hours, as indicated, and cell growth andsurvival was determined with the conventional MTT assay (control,non-treated cells were set at 100%). MTT assays were performed in96-well plates with the use of 3.0-8.0×10³ cells per well as describedin detail elsewhere (34). In parallel the expression levels of CHOP andGRP78 protein were determined by Western blot analysis. Note that DMC isthe most potent drug, celecoxib is substantially weaker, and none of theother coxibs or traditional NSAIDs are active under these conditions.

FIG. 9 shows that the knock-down of GRP78 enhances, whereas knock-downof Caspase 4 reduces, cell killing by celecoxib and DMC. U251glioblastoma cells were transiently transfected with si-RNA directed atGRP78 (si-GRP78) or caspase 4 (si-Casp 4). As a control, an siRNAtargeted at green fluorescent protein (si-GFP) was used. (A) Seventy-twohours after transfection, parallel cultures were treated with 40 μMcelecoxib (Cxb) and 30 μM DMC in the case of si-GRP78/si-GFP, or with 60μM Cxb and 40 μM DMC in the case of si-Casp 4/si-GFP; in all instances,control cultures received no drug treatment or treatment with solvent(DMSO) alone. After 48 hours of drug treatment, the drugs were removedand the fraction of surviving cells was determined by colony formingassays. Shown is the percentage of surviving cells (where the number ofcolonies under non-drug treated conditions was set to 100%). Thep-values shown demonstrate statistically significant differences insurvival between cells receiving si-GRP78 and control siRNA (si-GFP),and between cells receiving si-Casp 4 and control si-RNA, respectively.(B) In order to verify the effectiveness of the si-RNAs, Western blotanalysis of the target proteins was performed. Note that the knock-downof GRP78 leads to increased levels of CHOP protein, as expected from themodel of ER stress, where GRP78 signaling is upstream of CHOP. Caspase 4si-RNA also down-regulates its target (and cleaved caspase 4 becomesundetectable), but does not affect the induction of GRP78 in response tocelecoxib or DMC, as expected from the ER stress model, where caspase 4is downstream of GRP78 signaling.

FIG. 10 shows that DMC and celecoxib, but not rofecoxib, stimulate ERstress response and apoptosis in tumor cells in vivo. Nude mice wereimplanted subcutaneously with U87 glioblastoma cells. Once tumors hadreached a volume of 500 mm³, two animals each received either DMC orrofecoxib (150 mg/kg), or no drug for 36 hours. Thereafter, all sixanimals were sacrificed and their tumors analyzed by immunohistochemicalstaining for CHOP protein, as well as by TUNEL assay for celldeath/apoptosis. Left panels: expression of CHOP protein (small blackrectangles denote enlarged areas of the same photograph shown in themiddle panels). Right panels: cell death (arrows indicate examples ofTUNEL-positive, i.e., apoptotic, cells). The entire experiment wasrepeated with increasing daily dosages of drugs (including celecoxib)for 50 hours (see Materials & Methods), and similar results wereobtained. In all cases, representative sections are shown.

FIG. 11 shows that DMC, but not rofecoxib, inhibits tumor growth invivo. Nude mice were implanted subcutaneously with U87 glioblastomacells. Once palpable tumors had formed, the animals received daily chowsupplemented with DMC, rofecoxib, or no drug. Tumor size was determinedevery three days. Shown is the average (mean±SD) tumor volume in eachgroup (n=5). Asterisks (**): p<0.01 between control and DMC-treatedanimals on day 42.

FIG. 12 shows that bortezomib, celecoxib, and DMC reduce glioblastomacell survival. Cell growth and survival of various glioblastoma celllines was determined by MTT assay after 48 hours of culture in thepresence of increasing concentrations of (A) bortezomib (BZM), (B)celecoxib (CXB), or (C) 2,5-dimethyl-celecoxib (DMC). For comparisonpurposes to the known anti-multiple myeloma effects of bortezomib, theRPMI/8226 multiple myeloma cell line was included in (A).

FIG. 13 shows that DMC and bortezomib induce indicators of ER stress.U251 glioblastoma cells were cultured for 24 hours in the presence ofincreasing concentrations of (A) bortezomib (BZM) or (B) DMC. Total celllysates were analyzed by Western blot with specific antibodies toubiquitin, GRP78, CHOP, and caspase 4. Actin was used as a loadingcontrol. Pro-casp-4 denotes the inactive caspase 4 proenzyme, whereascleaved casp-4 is indicative of the activated form of the enzyme. Forcomparison purposes, cells treated with 10 nM bortezomib were analyzedside-by-side to DMC-treated cells in (B), indicating that the amount ofubiquitination is much more prevalent in bortezomib-treated cells thanin DMC-treated cells. Note that in the caspase 4 blots severalnon-specific background bands were observed (consistent with similarobservations in the literature); the specific bands were identified withthe use of various controls (not shown) and comparison to theliterature.

FIG. 14 shows that celecoxib and DMC enhance growth inhibition and celldeath by bortezomib. Glioblastoma cell lines were treated withbortezomib (BZM), celecoxib (CXB), or DMC alone and in combination. (A)Photomicrographs depicting the effects of combined drug treatment inLN229 and U251 cells after 48 hours of drug treatment. Representativesections are shown. (B) Quantitative analysis of combination drugeffects. LN229 cells were treated with drugs as above for 8, 24, and 48hours. Cell viability was determined by trypan blue exclusion assay. Theassays were performed with triplicate samples, and results arerepresentative of three independent experiments. Shown is the number ofviable cells under each condition (mean±SD). (C) U87MG cells werecultured for 24 hours in the presence of drugs as indicated, and theextent of cell death was determined by cell death ELISA (shown as meanpercent; n=4; ±SD). (D) LN229 cells were treated with drugs for 48 hoursand the number of long-term surviving cells that were able to spawn acolony was determined two weeks thereafter (colony formation assay).Shown is the percentage (mean±SD) of surviving cells from triplicateexperiments. The number of colonies obtained from non-drug treatedcontrols was set at 100%. In A-D, the following drug concentrations wereused: LN229: 5 nM BZM, 60 μM CXB, 40 μM DMC; U251: 10 nM BZM, 50 μM CXB,or 30 μM DMC; U87MG: 5 nM BZM, 50 μM CXB, 35 μM DMC. Asterisk indicatesthat the difference between individual drug treatments and combinationdrug treatments was statistically highly significant (p<0.001).

FIG. 15 shows that celecoxib and DMC enhance upregulation of indicatorsof ER stress and apoptosis by bortezomib. (A) U87MG and (B) T98G cellswere cultured in the presence of 10 nM bortezomib (BZM), 50 μM celecoxib(CXB), or 35 μM DMC individually or in combination as indicated for 24hours. Total cell lysates were analyzed by Western blot with specificantibodies to GRP78, CHOP, caspase-3 (Casp-3), caspase-4 (Casp-4),caspase-7 (Casp-7), caspase-9 (Casp-9), PARP and JNK, as indicated.Actin was used as a loading control. Pro-caspase denotes the full-length(inactive) caspase proenzymes, whereas cleaved caspases represent theactivated forms of these enzymes. The activity of JNK1 and JNK2 wasdetermined with the use of an antibody that specifically recognizes JNKphosphorylated on Thr183/Tyr185 (p-JNK1/2). Equal amounts of JNK1 wereconfirmed with an antibody that reacts with all JNK forms present.

FIG. 16 shows that knock-down of GRP78 enhances cell killing bycombination drug treatment. U251 cells were transiently transfected withsi-RNA directed at GRP78 (si-GRP78). As a control, an siRNA targeted atgreen fluorescent protein (si-GFP) was used. (A) Seventy-two hours aftertransfection, parallel cultures were treated with 5 nM bortezomib (BZM)combined with either 25 μM celecoxib (CXB) or 15 μM DMC. In parallel,transfected control cultures received no drug treatment or treatmentwith solvent (DMSO) alone. After 48 hours, the drugs were removed andthe fraction of surviving cells was determined by colony forming assaysover the course of 12-14 days. Shown is the percentage of survivingcells able to spawn a colony (where the number of colonies undernon-drug treated conditions was set to 100%). The reduction in colonynumbers in drug-treated siGRP78-transfected versus drug-treatedsiGFP-transfected cells was statistically significant (p<0.002). (B) Inorder to verify the effectiveness and specificity of the transfectedsi-RNAs, Western blot analysis of GRP78 expression was performed fromsiGFP-transfected and from siGRP78-transfected cells treated with therespective drugs in parallel. Both of these blots were processed anddeveloped simultaneously, and therefore can be directly comparedside-by-side. Although the down-regulation of GRP78 by its siRNA was not100% effective, the levels of this protein nonetheless were consistentlylowered in each condition as compared to the matching control cellstransfected with siGFP. Of note, overall lower concentrations of eachdrug were used in this experiment to enable the detection of furtherenhanced cell death by siRNA pretreatment.

FIG. 17 shows that DMC enhances bortezomib's effects on ER stress andapoptosis in vivo, Tumor-bearing mice were treated with 1 mg/kgbortezomib (BZM) and 7.5 mg/kg DMC individually or in combination, orremained untreated. Fifty hours later, the animals were sacrificed andthe tumors analyzed by immunohistochemical staining for CHOP (ER stressindicator) or by TUNEL (apoptosis indicator). (A) The top panels showtumor tissue stained with CHOP antibodies, and the middle panels showenlarged areas (indicated by the small rectangles) of the same sections.The bottom panels display TUNEL staining; a few select TUNEL-positivecells are indicated by arrows. (B) The percentage of TUNEL-positivecells (as indicated by reddish-brown stain) was determined in tenrandomly chosen microscopic fields from each treatment group and ispresented as mean±SD. Statistically significant differences in theextent of tumor cell death between individual and combination drugtreatments are indicated in the chart.

FIG. 18 shows the synergistic effect between Temozolomide (TMZ), thestandard of treatment for gliomas, and the non-coxib celecoxib analogue,2,5-dimethyl-celecoxib (DMC) that acts via aggravated ER Stress. Theresults show enhanced cell killing of tumor-associated brain endothelialcells (TuBEC) that were treated with a combination of TMZ (300 uM) and20 uM DMC for 48 hours. After drug treatment the cells were incubatedfor another 12 days in the absence of drug, in order to determine theirlong-term survival; at that time cytotoxicity was evaluated using thetrypan blue exclusion technique. Results are presented as number ofviable cells per group.

FIG. 19 shows the relative ability of three related derivatives inaccordance with embodiments of the present invention to induce apoptosisin human glioblastoma cells. The most potent compound was the2,5-ditrifluoromethyl derivative (DTF3C).

DETAILED DESCRIPTION

As set forth in the summary, the present invention is based on theunexpected discovery of a new strategy for using the ER-stress responsemechanism to induce apoptosis. In accordance with the strategy of thepresent invention, embodiments of the present invention provide methodsand tools for implementing the strategy. Specifically, the presentinvention provides a new class of chemotherapeutic compounds capable ofmodulating endoplasmic reticulum stress (ESR) that induces apoptosis(cell death) in both actively proliferating and quiescent cancer cells.These compounds also inhibit tumor invasion, vasculogenesis, andangiogenesis. Moreover, these compounds are characterized by highbioavailability (including the CNS), exhibit little toxicity, can beadministered orally, and may be combined with standard chemotherapeuticagents for enhanced anticancer effect. Therapeutic methods according tosome aspects of the present invention are aimed at treating cancer as aliving entity, not as a collection of aberrant cells, and may be appliedto all cancers.

Although not intending to be limited to any particular theory, a briefdiscussion of the ER stress response mechanism is provided herein tofacilitate a full and complete understanding of the strategy of thepresent invention.

Model of ER-Stress Response

The endoplasmic reticulum (ER) stress response (ESR) consists of a setof adaptive pathways that can be triggered by disparate perturbations ofnormal ER function, such as accumulation of unfolded proteins, lipid orglycolipid imbalances, or changes in the ionic conditions of the ERlumen (see (5, 6) for reviews). The primary purpose of the ESR is toalleviate stressful disturbance and restore proper ER homeostasis;however, in the case of intense or persistent ER stress, these pathwayswill trigger programmed cell death/apoptosis. One of the centralpro-survival regulators of the ESR is glucose-regulated protein 78(GRP78/BiP), which has important roles in protein folding and assembly,in targeting misfolded protein for degradation, in ER Ca²⁺-binding, andin controlling the activation of trans-membrane ER stress sensors (7).On the other hand, CCAAT/enhancer binding protein homologoustranscription factor (CHOP/GADD153) and caspase 4 are criticalexecutioners of the pro-apoptotic arm of the ESR (8, 9).

FIG. 1 illustrates a simplified model of ER stress response. In thisfigure, top-to-bottom alignment corresponds to the duration of stress(9). The different signaling events are grouped left-to-right accordingto whether they have a pro-apoptotic (apoptosis) or anti-apoptotic(survival) effect on the cell. The scale at the bottom of the figuresignifies the intricate balance between these two types of signals. Inan early phase of the stress response, translational attenuation occursto reduce the load of ER stress. In the next phase, several groups ofgenes are transcriptionally induced for long-term adaptation to ERstress. New synthesis of stress-induced proteins escapes from thegeneral translational attenuation. To cope with the unfolded proteins inthe ER, ER chaperones are first induced to refold them and if thisresponse is inadequate, ER associated degradation (ERAD) components arethen induced to eliminate the unfolded proteins. To remodel the ER, avariety of genes such as those of amino-acid import, glutathionebiosynthesis, and oxidation protection are also induced. To elicitimmune response and antiapoptotic effect, NFκB is activated. On theother hand, if severe ER stress conditions persist, the apoptosissignaling pathways are activated, including induction of CHOP andactivation of c-Jun N-terminal kinase (JNK kinase) and caspase-12. Thepivot point between survival or apoptosis may depend on the balancebetween survival signaling and apoptosis signaling.

FIG. 2 illustrates the three conditions of ER stress, i.e. no ER stress,low ER stress and severe ER stress. The present invention describesmethods, compounds and compositions which are capable of inducingapoptosis in those cells that are in a state of low level ER stress butare otherwise able to survive. The provided compounds “nudge” such cellswith low ER stress towards a severe ER stress condition that pivots thesignaling balance of the cell away from survivable conditions andtowards apoptosis. The ability of the provided compounds to simplyaggravate ER stress allows their use in this manner even if they exhibitonly moderate pro-apoptotic potency, which also minimizes the inductionof apoptosis in normal cells that typically exist under conditions of noER stress. Under typical low ER stress conditions GRP78 is expressed inthe cells at levels that are typically greater than two times itsoccurrence in normal cells while the corresponding levels of CHOP arenot sufficient to initiate apoptosis. However, upon the use of compoundsand compositions provided by this invention, the levels of CHOP areincreased to levels sufficient to overcome the protective effects of thecorresponding levels of GRP78, resulting in a condition of severe stressand the initiation of apoptosis. In essence the provided compounds,rather than being able to initiate ER stress and induce apoptosis in anytype of cells, simply serve as “the straw that breaks the camel's back”by aggravating the ER stress of cells that are already in low ER stress.This key feature of the provided compounds enables their ability toserve as well-tolerated potential therapeutic agents for a variety ofconditions whose pathogenesis involves cells in a state of low ER stressthat can benefit from the induction of apoptosis in such cells. Thissituation exists in many forms of cancer as well as several otherdiseases.

ER Stress Response and Cancer Therapy

Although the relevance of ER stress to tumor growth and survival hasbegun to be recognized, very little is known with regards to itspotential exploitation for purposes of tumor therapy. More importantly,there are yet no known points of attack in the ER stress responsepathway or compounds that may provide a basis for viable therapeuticregimens.

For example, one potentially effective mode of triggering ER stressresponse is the inhibition of the sarcoplasmic/endoplasmic reticulumCa²⁺-ATPase (SERCA), an intracellular membrane bound enzyme whichsequesters cytosolic Ca²⁺ into its intracellular ER storage compartment.Inhibition of SERCA leads to the release of Ca²⁺ into the cytoplasm andresulting in activation of severe ER stress, leading to apoptosis.Although several inhibitors of SERCA have been described, none of thesehas an acceptable therapeutic profile to be used as a proapoptotic agentfor cancer therapy. For instance, the most potent SERCA inhibitor, thenatural product thapsigargin, is not suitable as a therapeutic agent dueto its high toxicity and its histamine-release ability.

Another compound that was found to be a SERCA inhibitor is theanti-inflammatory drug celecoxib (Celebrex®) which is an inhibitor ofcyclooxygenases-2 (COX-2) (Dannenberg, A J and Subbaramaiah, K, CancerCell 2003:4:431). However, long-term use of COX-2 inhibitors has beenlinked to potentially life-threatening cardiovascular risks, thatresulted in the withdrawal of the drug rofecoxib (Vioxx®) (Funk, C D andFitzgerald, G A, Journal of Cardiovascular Pharmacology 2007; 50:470).Moreover, it is generally believed that the biochemical mechanismunderlying the anticancer activities of COX-2 inhibitors (coxibs) andother NSAIDs are through the inhibition of cyclooxygenase (COX) enzymes,which catalyze the initial step in prostaglandin synthesis (10). Thefact that celecoxib is a member of the COX-2 specific inhibitors knownas coxibs leaves open the possibility that its anticancer activity canbe also linked to COX-2 inhibition.

In several recent studies, it has been noted that treatment of culturedcells with various NSAIDs, including celecoxib, generated increasedlevels of intracellular calcium ([Ca²]_(i)) with subsequent activationof the ER stress response (ref. 11-16). Although these observations hintat the possibility that NSAID, as a class, may induce apoptosis throughthe ER stress response pathway, the high level of NSAIDs concentration(0.1 to >1.0 mmol/L) needed to elicit this effect makes such casualconnection highly speculative. At these concentrations, other effects ofNSAIDs such as COX-2 inhibition will likely dominate, rendering themunlikely candidates as a tool for inducing apoptosis with highspecificity.

Surprisingly, in a series of reports, it was found that celecoxib canstill exert potent anti-proliferative and pro-apoptotic effects in theabsence of any apparent involvement of COX-2 (ref 17-23). While theseobservations suggest that celecoxib may have a secondary targetindependent of COX-2 which leads to apoptosis in the cancerous cells,the underlying mechanism was not understood.

The inventors of the present invention have undertaken experiments tostudy this unknown mechanism and have unexpectedly discovered that theCOX-2 independent antitumor activity of celecoxib is in fact through theER stress response pathway. Moreover, it was found that there existstructural analogues of celecoxib which exhibit potent activity ininducing ER stress, both in vitro and in vivo (see EXAMPLES forsupporting experiments and data). Although NSAIDs are known to be ableto stimulate ER stress response at high concentrations and triggeringthe expression of both the prosurvival GRP78 and the proapoptotic CHOPproteins, apparently, the effect of celecoxib resulted in intense ERstress which led to elevated CHOP expression that overwhelmed theprotective effect of GRP78, which in turn activated caspase-4 andcommitted the cell to apoptosis.

DEFINITIONS

As used herein, the phrase “ER stress” collectively refers to thevarious physiological and pathological conditions that may impairprotein synthesis in the ER.

As used herein, the term “SERCA” is an acronym forSarcoplasmic/Endoplasmic Reticulum Ca²⁺-ATPase, which is a small familyof highly conserved proteins (isoforms), all of which function ascalcium transmembrane pumps in a similar manner.

As used herein, the term “COX-2” refers to cyclooxygenase-2, also knownas prostaglandin-endoperoxidase 2 (prostaglandin G/H synthase andcyclooxygenase)

As used herein, the term “CHOP” refers to CCAAT/enhancer binding proteinhomologous transcription factor, also called growth arrest and DNAdamage-inducible gene 153 (GADD153)

As used herein, the phrase “ER-stress aggravating agent” refers to anagent that is capable of inducing or aggravating a stress in ER, whetherdirectly or indirectly. The constitution of such an agent is notparticularly limited, and may be chemical or physical in nature.Exemplary physical agents may include temperature, radiation, and sound,but not limited thereto. Exemplary chemical agents may include cellularsignaling molecules, cytotoxic agents, toxins, or other metabolites, butnot limited thereto In addition, specific conditions of the cellularmicroenvironment, such as hypoxia, low pH, shortage of nutrients, maytrigger ER stress.

As used herein, the term “prodrug” refers to a pharmacological substance(drug) where the parent molecule is either inactive or minimal activity.

As used herein, the term “synergy” or “synergistic effect” refer to drugcombinations where the resultant pharmacologic effect is greater thantwo times the activity compare with the activity of the drugs usedindividually (i.e. the combination is greater than the sum of itsparts).

As used herein, the nomenclature alkyl, alkoxy, carbonyl, etc. is usedas is generally understood by those of skill in this art.

As used in this specification, alkyl groups can includestraight-chained, branched and cyclic alkyl radicals containing up toabout 20 carbons, or 1 to 16 carbons, and are straight or branched.Exemplary alkyl groups herein include, but are not limited to, methyl,ethyl, propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl,isopentyl, neopentyl, tert-pentyl and isohexyl. As used herein, loweralkyl refer to carbon chains having from about 1 or about 2 carbons upto about 6 carbons. Suitable alkyl groups may be saturated orunsaturated. Further, an alkyl may also be substituted one or more timeson one or more carbons with substituents selected from a groupconsisting of C1-C15 alkyl, allyl, allenyl, alkenyl, C₃-C₇ heterocycle,aryl, halo, hydroxy, amino, cyano, oxo, thio, alkoxy, formyl, carboxy,carboxamido, phosphoryl, phosphonate, phosphonamido, sulfonyl,alkylsulfonate, arylsulfonate, and sulfonamide. Additionally, an alkylgroup may contain up to 10 heteroatoms, in certain embodiments, 1, 2, 3,4, 5, 6, 7, 8 or 9 heteroatom substituents. Suitable heteroatoms includenitrogen, oxygen, sulfur and phosphorous.

As used herein, “cycloalkyl” refers to a mono- or multicyclic ringsystem, in certain embodiments of 3 to 10 carbon atoms, in otherembodiments of 3 to 6 carbon atoms. The ring systems of the cycloalkylgroup may be composed of one ring or two or more rings which may bejoined together in a fused, bridged or spiro-connected fashion.

As used herein, “aryl” refers to aromatic monocyclic or multicyclicgroups containing from 3 to 16 carbon atoms. As used in thisspecification, aryl groups are aryl radicals which may contain up to 10heteroatoms, in certain embodiments, 1, 2, 3 or 4 heteroatoms. An arylgroup may also be optionally substituted one or more times, in certainembodiments, 1 to 3 or 4 times with an aryl group or a lower alkyl groupand it may be also fused to other aryl or cycloalkyl rings. Suitablearyl groups include, for example, phenyl, naphthyl, tolyl, imidazolyl,pyridyl, pyrroyl, thienyl, pyrimidyl, thiazolyl and furyl groups.

As used in this specification, a ring is defined as having up to 20atoms that may include one or more nitrogen, oxygen, sulfur orphosphorous atoms, provided that the ring can have one or moresubstituents selected from the group consisting of hydrogen, alkyl,allyl, alkenyl, alkynyl, aryl, heteroaryl, chloro, iodo, bromo, fluoro,hydroxy, alkoxy, aryloxy, carboxy, amino, alkylamino, dialkylamino,acylamino, carboxamido, cyano, oxo, thio, alkylthio, arylthio, acylthio,alkylsulfonate, arylsulfonate, phosphoryl, phosphonate, phosphonamido,and sulfonyl, and further provided that the ring may also contain one ormore fused rings, including carbocyclic, heterocyclic, aryl orheteroaryl rings.

As used herein, alkenyl and alkynyl carbon chains, if not specified,contain from 2 to 20 carbons, or 2 to 16 carbons, and are straight orbranched. Alkenyl carbon chains of from 2 to 20 carbons, in certainembodiments, contain 1 to 8 double bonds, and the alkenyl carbon chainsof 2 to 16 carbons, in certain embodiments, contain 1 to 5 double bonds.Alkynyl carbon chains of from 2 to 20 carbons, in certain embodiments,contain 1 to 8 triple bonds, and the alkynyl carbon chains of 2 to 16carbons, in certain embodiments, contain 1 to 5 triple bonds.

As used herein, “heteroaryl” refers to a monocyclic or multicyclicaromatic ring system, in certain embodiments, of about 5 to about 15members where one or more, in one embodiment 1 to 3, of the atoms in thering system is a heteroatom, that is, an element other than carbon,including but not limited to, nitrogen, oxygen or sulfur. The heteroarylgroup may be optionally fused to a benzene ring. Heteroaryl groupsinclude, but are not limited to, furyl, imidazolyl, pyrrolidinyl,pyrimidinyl, tetrazolyl, thienyl, pyridyl, pyrrolyl, N-methylpyrrolyl,quinolinyl and isoquinolinyl.

As used herein, “heterocyclyl” refers to a monocyclic or multicyclicnon-aromatic ring system, in one embodiment of 3 to 10 members, inanother embodiment of 4 to 7 members, in a further embodiment of 5 to 6members, where one or more, in certain embodiments, 1 to 3, of the atomsin the ring system is a heteroatom, that is, an element other thancarbon, including but not limited to, nitrogen, oxygen or sulfur. Inembodiments where the heteroatom(s) is(are) nitrogen, the nitrogen isoptionally substituted with alkyl, alkenyl, alkynyl, aryl, heteroaryl,aralkyl, heteroaralkyl, cycloalkyl, heterocyclyl, cycloalkylalkyl,heterocyclylalkyl, acyl, guanidino, or the nitrogen may be quaternizedto form an ammonium group where the substituents are selected as above.

EXEMPLARY EMBODIMENTS OF THE PRESENT INVENTION 1. Methods for Inducingor Aggravating Stress in a Cell's ER to Trigger Apoptosis

In a first aspect, the present invention provides a method of inducingor aggravating stress in a cell's endoplasmic reticulum (ER) to triggerapoptosis.

As set forth above, methods according to this aspect of the presentinvention are based on the discovery that by inducing or aggravating ERfor a sufficient intensity and duration, the proapoptotic effect of theelevated expression of CHOP will overtake the protective effect ofGRP78, leading to activation of a caspase such as caspase 4 and/orcaspase 7, thereby, committing the cell to apoptosis. It is also basedon the discovery that there exist agents capable of specificallyinducing or aggravating ER stress in a selected cell without causingconcomitant side-effects in other non-targeted cells.

Specifically, preferred embodiments in accordance with this aspect ofthe present invention generally comprise the steps of 1). inhibitingSERCA activity selectively in said cell without inhibiting COX-2activity; and 2). elevating the expression of CHOP in said cell,wherein, the combination of inhibiting SERCA activity and elevating CHOPexpression results in a condition favorable for initiation of apoptosisin the cell.

It is envisioned that aggravation of ER stress via the inhibition ofSERCA can be achieved with any suitable agent known in the art or futureidentified agents that are developed to inhibit SERCA and induceapoptosis.

Preferably, the specificity of the agent is such that it does notinhibit COX-2 activity or trigger the release of histamine.

In a preferred embodiment, selective inhibition of SERCA activity isaccomplished by administering a pharmaceutically effective amount of anER-stress aggravating agent capable of selectively inhibiting SERCA,directly or indirectly, for a duration sufficient to elevate cytoplasmiccalcium concentration significantly above normal level so as to causeapoptosis. In this embodiment, elevation of CHOP expression may beco-effected as a result of the aggravated ER-stress, or may bespecifically induced by a CHOP expression enhancer/inducer. ExemplaryCHOP expression enhancer/inducer may include transfection ofplasmid-based constructs or infection with viral expression constructs,but is not limited thereto.

1.1 Embodiments in Which the ER-Stress Aggravating Agent is a Compoundor a Pharmaceutical Composition

In some preferred embodiments, the ER-stress aggravating agent is acompound or a pharmaceutical composition containing the compound,wherein the compound is one having the general formula:

and wherein:

-   R₁ is methyl, fluoromethyl, difluoromethyl, trifluoromethyl, alkyl,    fluoroalkyl, difluoroalkyl, trifluoroalkyl, polyfluoroalkyl,    hydroxyalkyl, or carboxyalkyl;-   R₂ is hydrogen, fluoro, chloro, bromo; fluoromethyl, difluoromethyl,    trifluoromethyl, alkyl, fluoroalkyl, difluoroalkyl, trifluoroalkyl,    polyfluoroalkyl, hydroxyalkyl, or carboxyalkyl;-   R₃-R₇ are independently selected from a group consisting of:    hydrogen, fluoro, chloro, bromo, alloxy, alkyl, fluoroalkyl,    difluoroalkyl, trifluoroalkyl, polyfluoroalkyl, hydroxyalkyl, or    carboxyalkyl, aryl and heteroaryl;-   R₈-R₁₁ are independently selected from a group consisting of:    hydrogen, fluoro, chloro, bromo; fluoromethyl, difluoromethyl,    trifluoromethyl, alkyl, fluoroalkyl, difluoroalkyl, trifluoroalkyl,    polyfluoroalkyl, hydroxyalkyl, carboxyalkyl, alkenyl, alkynyl,    cycloalkyl, heterocyclic, aryl, or heteroaryl; and-   R₁₂ is hydrogen, acetyl, acyl, alkyl, fluoroalkyl, difluoroalkyl,    trifluoroalkyl, polyfluoroalkyl, hydroxyalkyl, carboxyalkyl;    aminoacyl, aminoalkyl, cycloalkyl, heterocyclic, aryl, or    heteroaryl.

In some more preferred embodiments, R₁ is trifluoromethyl.

In other preferred embodiments, R₂, R₈-R₁₂ are hydrogen, or R₄, R₅ andR₇ are hydrogen. In one embodiment, R₁ is trifluoromethyl and R₂, R₄,R₅, R₇-R₁₂ are all hydrogen.

In another preferred embodiment, the aggravating agent is4-[5-(2,5-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide,4-[5-(2,5-di(trifluoromethyl)phenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide,4-[5-(2,5-dibromophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide,or an analogue thereof.

For compounds that contain at least one fluorine-containing functionalgroup, such as a CF3 group, the provided compounds can enable themonitoring and imaging of their in vivo actions by using magneticresonance imaging. This can be a very valuable feature for difficult totreat and difficult to monitor diseases such as brain cancer.

1.2 Embodiments in Which the Aggravating Agent is a Prodrug

As set forth above, the present invention also includes thoseembodiments in which either directly or indirectly inhibits SERCA andthus aggravating ER stress. In one exemplary embodiment, the aggravatingagent is a prodrug which can be converted into an active compound ableto inhibit SERCA via metabolism or other conversion means. For example,in those embodiments where the aggravating agent is a recombinantmolecule, a translocation peptide may be attached to allow the agent totranslocate across the membrane. In other embodiments the providedcompounds contain functional groups that upon metabolic oxidation,proteolytic cleavage or hydrolysis convert the provided compound into anactive ER aggravating agent. Those skilled in the art can recognize thatsuch functional groups include but are not limited to methyl or alkylgroups, ester groups, carbonate groups, amide groups, peptides, or theircyclic or polymeric derivatives.

1.3 Embodiments in Which a Combination of Aggravating Agent is Used

Methods in accordance with some embodiments of the present invention mayfurther include a step of applying an additional one or more ER-stressaggravating agent. Preferably, this additional one or more ER-stressaggravating agent acts through a different stress-inducing mechanism.

It is another unexpected discovery of the present invention that whendifferent strategies of aggravating ER-stress are used in combination, asynergistic effect may be achieved.

For example, in one embodiment, when the drug bortezomib (which inducesapoptosis through inhibition of proteasome 26S) is used together withcelecoxib, the level of ER-stress was elevated and the rate of apoptosiswas significantly increased (see Example 2).

Accordingly, it is envisioned that any of the apoptosis inductionstrategies listed in Table 1 may be advantageously combined withstrategies of the present invention (i.e. induction of apoptosis throughER-stress aggravation) to achieve a synergistic effect in inducingapoptosis.

In some preferred embodiments, a first ER-stress aggravating agentcapable of selectively inhibiting SERCA activity without inhibitingCOX-2 or triggering histamine release may be combined with a secondER-stress aggravating agent which is capable of increasing theconcentration of misfolded or damaged protein in the ER. Exemplaryagents suitable for use as the second ER-stress aggravating agent mayinclude a proteasome inhibitor (e.g. bortezomib, or an analoguethereof), or a protease inhibitor (e.g. nelfinavir, atazanavir,fosamprenavir, ritonavir, indinavir, or an analogue thereof).

1.4 Embodiments in Which an Apoptosis Enhancer is Used

In some further embodiments of the present invention, the ER-stressaggravating agent may be assisted by further applying an apoptosisenhancer to the cell. In the context of the present invention, anapoptosis enhancer is one that amplifies the effect of ER-stress inrelation to the induction of apoptosis. Preferably, an apoptosisenhancer is a chemical or biological agent that is capable ofupregulating the expression of CHOP, overcoming the protective effect ofGRP78, and activating a caspase such as caspase-4 and/or caspase-7.Exemplary apoptosis enhancer may include a siRNA for GRP78 or aninhibitor of GRP78 function, but not limited thereto. Additionally,apoptosis enhancers may also be synthetic or natural compounds,including, but not limited to, BH3-mimetics that block the activity ofanti-apoptotic proteins of the Bcl-2 family, such as ABT-737.

2. Method for Screening, Selecting, or Designing Compounds Useful as anER-Stress Aggravating Agent

In a second aspect, the present invention provides a method forscreening, selecting, or designing compounds useful for inducing oraggravating ER stress in a cell to trigger apoptosis in the cell.

Embodiments according to this aspect of the present invention have thegeneral steps of: 1). Obtaining information about a test compound; 2).Identifying the test compound as a potential ER-stress agent if the testcompound is an inhibitor of SERCA and is not an inhibitor of COX-2.Preferably, the identified compound also does not trigger the release ofhistamine. In the obtaining step, information about the test compound tobe obtained may include SERCA inhibitory activity of the compound, COX-2inhibitory activity of the compound, and histamine release activity ofthe compound.

The evidence of ER stress in a cell can be determined by the presence ofhigh levels of GRP78 that occur at levels greater than twice the GRP78levels in normal tissues. The levels of GRP78 can be determined by thoseskilled in the art using common techniques, such as those described inthe examples.

Source of the test compound may come from any common source known in theart, including commercial chemical vendors, chemical libraries generatedby combinatorial chemistry, or modified analogues of known SERCAinhibitors, but are not limited thereto.

In the case where the desired information about a test compound is notalready known or otherwise available, the step of obtaining informationmay involve performing assays to characterize the test compounds. Insuch cases, any assays commonly known in the art may be used, includingchemical assays and cell-based assays, but not limited thereto.Preferably, the assays chosen should yield quantifiable results easilycomparable to other test compounds.

Because screening of new compounds is necessarily a trial-and-errorprocess and the steps of obtaining information, identifying thepromising candidate compounds from the test compounds are repeated foreach test compound.

In some preferred embodiments, a large number of test compounds are usedand the steps of obtaining, identifying, and repeating are performed ina high-throughput format. General principles of high-throughput drugscreening are well-known in the art (for a recent review, see Walters etal., Nat Rev Drug Discov. 2003 April; 2(4):259-66, the content of whichis incorporated herein by reference).

In addition to screening existing chemical sources, once a sufficientnumber of compounds have been tested and identified, computationalanalysis may also be employed to further optimize or generate newcandidate compounds suitable for use as an ER-stress aggravating agent.Any commonly known computational drug discovery methods may beadvantageously adapted for this task. Preferably, ligand-basedmethodologies are used. (for a recent review on ligand-based drugdesign, see Bacilieri et al., Current Drug Discovery Technologies,Volume 3, Number 3, September 2006, pp. 155-165(11), the content ofwhich is incorporated herein by reference).

In a preferred embodiment, a quantitative structure-activityrelationship (QSAR) analysis is performed (for a review on adaptationand incorporation of computational methodologies with high-throughputscreening, see Davies et al., Curr Opin Chem Biol. 2006 August;10(4):343-51. Epub 2006 Jul. 5, the content of which is incorporatedwherein by reference).

3. Compounds Useful as an ER-Stress Aggravating Agent

In a third aspect, the present invention also provides compounds usefulas an ER stress aggravating agent, having the general formula:

wherein,

-   R₁ is methyl, fluoromethyl, difluoromethyl, trifluoromethyl, alkyl,    fluoroalkyl, difluoroalkyl, trifluoroalkyl, polyfluoroalkyl,    hydroxyalkyl, or carboxyalkyl;-   R₂ is hydrogen, fluoro, chloro, bromo; fluoromethyl, difluoromethyl,    trifluoromethyl, alkyl, fluoroalkyl, difluoroalkyl, trifluoroalkyl,    polyfluoroalkyl, hydroxyalkyl, or carboxyalkyl;-   R₃-R₇ are independently selected from a group consisting of:    hydrogen, fluoro, chloro, bromo, alloxy, alkyl, fluoroalkyl,    difluoroalkyl, trifluoroalkyl, polyfluoroalkyl, hydroxyalkyl, or    carboxyalkyl, aryl and heteroaryl;-   R₅-R₁₁ are independently selected from a group consisting of:    hydrogen, fluoro, chloro, bromo; fluoromethyl, difluoromethyl,    trifluoromethyl, alkyl, fluoroalkyl, difluoroalkyl, trifluoroalkyl,    polyfluoroalkyl, hydroxyalkyl, carboxyalkyl, alkenyl, alkynyl,    cycloalkyl, heterocyclic, aryl, or heteroaryl; and-   R₁₂ is hydrogen, acetyl, acyl, alkyl, fluoroalkyl, difluoroalkyl,    trifluoroalkyl, polyfluoroalkyl, hydroxyalkyl, carboxyalkyl;    aminoacyl, aminoalkyl, cycloalkyl, heterocyclic, aryl, or    heteroaryl.

Preferred embodiments are compounds wherein R₁ is trifluoromethyl, whileother preferred embodiments are compounds wherein R₂, R₈ and R₉ arehydrogen or compounds wherein R₄, R₅, and R₇ are hydrogen, as well ascompounds wherein R₁ is trifluoromethyl, and R₂, R₄, R₅, R₇, R₈ and R₉are hydrogen.

Other preferred embodiments are compounds wherein R₃ and R₆ are selectedfrom a group consisting of: hydrogen, fluoro, chloro, bromo;fluoromethyl, difluoromethyl, trifluoromethyl, alkyl, aryl, heteroaryl,fluoroalkyl, difluoroalkyl, trifluoroalkyl, polyfluoroalkyl,hydroxyalkyl, or carboxyalkyl.

An example of preferred embodiments are compounds that are structuralanalogs of4-[5-(2,5-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamidesuch as:4-[5-(2,5-di(trifluoromethyl)phenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamideand4-[5-(2,5-dibromophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide.

Referring to FIG. 19, two exemplary compounds in accordance with thepresent invention have shown surprisingly potent cell killing effectcompare to DMC. In the figure, DMC stands for4-[5-(2,5-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide;DBrC stands for4-[5-(2,5-dibromophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide;and DTF3C stands for4-[5-(2,5-ditrifluoromethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide.

Another preferred embodiment, the compounds are prodrug compounds thatcan be converted in vivo to an ER-stress aggravating agent that can actas a SERCA inhibitor. Those in the art will recognize such compounds ashaving the general structure:

wherein at least one group of R₁-R₁₂ has a side-chain selected from thelinker groups shown above.

4. Pharmaceutical Compositions Useful as an ER-Stress Aggravating Agent

In a fourth aspect, the present invention also provides a pharmaceuticalcomposition useful for inducing or aggravating ER stress in a cell totrigger apoptosis in the cell. Embodiments of the present inventiongenerally include an ER-stress aggravating agent, and a pharmaceuticallyacceptable carrier.

Any chemical-based ER-stress aggravating agent described above may besuitably formulated to facilitate administration of the activeingredient. In a preferred embodiment, the ER-stress aggravating agentis a compound having the general formula also described in the firstaspect above, or a pharmaceutically stable salt thereof.

For the carrier, any commonly known carrier that is compatible with thechosen active ingredient may be suitably used. Preferably, the carrieris one suitable for oral formulation.

5. Therapeutic Methods

In a fifth aspect, the present invention also provides a method fortreating a diseased condition in a patient by inducing or aggravating ERstress in selected diseased cells to trigger apoptosis in the cells.

Embodiments according to this aspect of the present invention includethe general steps of administering a pharmaceutically effective amountof a pharmaceutical composition according to the fourth aspect of thepresent invention described above. Any methods for inducing oraggravating ER stress according to embodiments set forth in the firstaspect of the present invention may be suitably adapted for treatmentmethods of the present invention.

In a preferred embodiment, the diseased cells are in a state of ERstress prior to administration of the pharmaceutical composition.

The method of treatment described herein is a general method that isapplicable to all forms of diseases whether in the pathogenesis includedisregulation of apoptosis as a contributing mechanism. In preferredembodiments, cancer is the preferred disease for the treatment methodsof the present invention. Exemplary cancers that may be applicableinclude glioblastoma multiforme, breast carcinoma, pancreatic carcinoma,Burkitt's lymphoma, multiple myeloma, neuroblastoma, prostate cancer,colorectal cancer, metastatic breast cancer, recurring cancer, anddrug-resistant cancer, but are not limited thereto.

Another embodiment of the presence invention involves the treatment ofchemoresistant tumors by inhibiting invasion, vasculogenesis, andangiogenesis. In chemoresistant tumor cells, the tumor vasculature isnot responsive to many therapeutic drugs, including Temozolomide (TMZ),the agent most commonly used for glioblastoma multiforme (GBM) therapy.The tumor vasculature provides the nutrients, oxygen as well as theenvironment for tumor growth. Therefore agents that destroy these cellswould be very useful in treating the residual or recurring tumors.

In yet another embodiment the present invention provides a method forthe treatment of the perinecrotic rim of cancers, involving theinduction of apoptosis via ER stress response modulation alone or incombination with conventional chemotherapy. More particularly, thisinvention can be employed for the treatment of especiallydifficult-to-treat cancers that do not respond well to chemotherapy,such as glioblastoma multiforme (GBM). Many cancers will outgrow theirblood supply, where the consequence is the development of a necroticcenter. The cancer cells at the “necrotic rim” are usually quiescentcells, exposed to an adverse microenvironment of low oxygenation, lowpH, and low glucose. As consequence of this stressful environment, thesecells have a high level of ESR. To thrive in this hostile environment,high resistance level of GRP78 is expressed to survive. Theseperinecrotic cancer cells are especially resistant to treatment bychemotherapy or radiation therapy, which typically target rapidlyproliferating cells.

The present invention offers unique benefits for the treatment of braintumors that are not possible with conventional chemotherapy. The bloodbrain barrier, although generally not intact at the blood-tumorinterface, still impedes free access of chemotherapy to the tumor.Moreover, traditional cytotoxic chemotherapy targets rapidlyproliferating cancer cells. GBM, however, is heterogeneous in nature.Pathologically, GBM is characterized by a necrotic center, aperinecrotic rim, and an invading vascularized periphery. Thisperinecrotic rim has been pathologically described as thepseudopalisades seen in pathological specimens in glioblastoma (Brat DJ, et al, Cancer Res 2004, 64: 920-7). Chemotherapy and radiationtraditionally target the well vascularized proliferating tumor cells inthe interface of normal brain and tumor tissue. In contrast, themalignant cells in the perinecrotic rim of the brain tumor undergo lessproliferation, and remain dormant, albeit with the potential to beactivated. Once the proliferating tumor cells are killed by chemotherapyor radiation, the dormant glioblastoma cells in the perinecrotic zonebegin to grow and invade again, repeating the process. This mechanism ofdormant cell activation is very similar to the concept of trees growingin a rainforest. A rainforest has trees of varying height. If thetallest trees (similar to the well vascularized tumor region) are cutdown, then the lower level trees (similar to the perinecrotic regioncells) will grow up as they now receive the bulk of the sunlight. Theend result is that the GBM recurs, with increased invasion into thenormal brain. The glioblastoma cells in the perinecrotic zone have beenpreviously characterized. They secrete increased levels of proteins inresponse to the low oxygen tension that is present in the perinecroticzone, and angiogenesis factors. The perinecrotic cells proliferatesignificantly less and exhibit more cell death than the glioblastomacells in the well vascularized periphery. Moreover, these tumor cellsdisplay increased migration, and increased secretion of specific enzymesassociated with cell invasion (Brat D J, et al., Cancer Res 2004, 64:920-7, the entire content of which is incorporated herein by reference).

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXAMPLES Example 1 Calcium-Activated ER Stress as a Major Component ofTumor Cell Death Induced by 2,5-dimethyl-celecoxib (DMC), a Non-CoxibAnalog of Celecoxib Materials and Methods Materials

Celecoxib is4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide(24). DMC is a close structural analogue, where the 5-aryl moiety hasbeen altered by replacing 4-methylphenyl with 2,5-dimethylphenyl,resulting in4-[5-(2,5-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide(20, 19). Both compounds were synthesized in our laboratory according topreviously published procedures (see ref. 24 for celecoxib and ref 19for DMC). Each drug was dissolved in DMSO at 100 mmol/L (stocksolution). For valdecoxib (25) and rofecoxib (26), commercial caplets ofBextra (Pfizer, New York, N.Y.) and Vioxx (Merck, Whitehouse Station,N.J.), respectively, were suspended in H2O to disintegrate theexcipient, and the active ingredient was dissolved in DMSO at 25 mmol/L.In addition, we used pure rofecoxib powder that was synthesized in ourlaboratory according to established procedures (27). All traditionalNSAIDs were purchased from Sigma (St. Louis, Mo.) in powdered form anddissolved in DMSO at 100 mmol/L. Thapsigargin and BAPTA-AM were obtainedfrom Sigma and dissolved in DMSO. All drugs were added to the cellculture medium in a manner that kept the final concentration of solvent(DMSO)<0.5%.

Cell Lines and Culture Conditions

Most cell lines were obtained from the American Type Culture Collection(Manassas, Va.) and were propagated in DMEM or RPMI 1640 (LifeTechnologies, Grand Island, N.Y.) supplemented with 10% fetal bovineserum, 160 units/mL penicillin, and 0.1 mg/mL streptomycin in ahumidified incubator at 37° C. and a 5% CO₂ atmosphere. The glioblastomacell lines U251 and LN229 were provided by Frank B. Fumari and WebsterK. Cavenee (Ludwig Institute of Cancer Research, La Jolla, Calif.).

Immunoblots and Antibodies

Total cell lysates were prepared by lysis of cells withradioimmunoprecipitation assay buffer (28), and protein concentrationswere determined using the bicinchoninic acid protein assay reagent(Pierce, Rockford, Ill.). For Western blot analysis, 50 Ag of eachsample were processed as described (29). The primary antibodies werepurchased from Cell Signaling Technologies (Beverly, Mass.), CaymanChemical (Ann Arbor, Mich.), or Santa Cruz Biotechnology, Inc. (SantaCruz, Calif.) and used according to the manufacturer's recommendations.The secondary antibodies were coupled to horseradish peroxidase anddetected by chemiluminescence using the SuperSignal West substrate fromPierce. All immunoblots were repeated at least once to confirm theresults.

Immunohistochemistry

Immunohistochemical analysis of protein expression in tumor tissues wasdone with the use of the Vectastain avidin-biotin complex method kit(Vector Laboratories, Burlingame, Calif.) according to themanufacturer's instructions. This procedure uses biotinylated secondaryantibodies and a preformed avidin: biotinylated enzyme complex that hasbeen termed the avidin-biotin complex method technique. As the primaryantibody, we used anti-CHOP antibody (Santa Cruz Biotechnology) diluted1:100 in 2% normal goat blocking serum.

Apoptosis Measurements

Apoptosis in tumor sections was measured quantitatively with the use ofthe terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling assay (30). All components for this procedure were from theApopTag In situ Apoptosis Detection kit (Chemicon, Temecula, Calif.),which was used according to the manufacturer's instructions.

Apoptosis in cell cultures in vitro was determined by using the CellDeath Detection ELISA kit (Roche Diagnostics, Indianapolis, Ind.)according to the manufacturer's instructions. This immunoassayspecifically detects the histone region (H1, H2A, H₂B, H3, and H4) ofmononucleosomes and oligonucleosomes that are released during apoptosis.Ninety-six-well plates were seeded with 1,000 cells per well and read at405 nm in a Microplate Autoreader (Model EL 311 SX; Bio-Tek Instruments,Inc., Winooski, Vt.).

Colony Formation Assay

Twenty-four hours after transfection with small interfering RNA (siRNA),the cells were seeded into six-well plates at 200 cells per well. Aftercomplete cell adherence, the cells were exposed to drug treatment for 48h. Thereafter, the drug was removed, fresh growth medium was added, andthe cells were kept in culture undisturbed for 12 to 14 days, duringwhich time the surviving cells spawned a colony of proliferating cells.Colonies were visualized by staining for 4 h with 1% methylene blue (inmethanol) and then counted.

Transfections with SiRNA

Cells were transfected in six-well plates with the use of LipofectAMINE2000 (Invitrogen, Carlsbad, Calif.) according to the manufacturer'sinstructions. The different siRNAs were synthesized at the microchemicalcore laboratory of the University of Southern California/K. Norris Jr.Comprehensive Cancer Center, and their sequences were as follows: siRNAtargeted at green fluorescent protein (si-GFP),5′-CAAGCUGACCCUGAAGUUCTT-3′ (sense) and 5′-GAACUUCAGGGUCAGCUUGTT-3′(antisense); si-GRP78, 5′-GGAGCGCAUUGAUACUAGATT-3′ (sense) and5′-UCUAGUAUCAAUGCGCUCCTT-3′ (antisense); and si-caspase-4,5′-AAGUGGCCUCUUCACAGUCAUTT-3′ (sense) and 5′-AAAUGACUGUGAAGAGGCCACTT-3′(antisense).

Cytoplasmic Calcium Imaging

The cells were loaded by incubating them with 4 Amol/L Fura-2/AM(Invitrogen) for 30 min at room temperature in external solutioncontaining 138 mmol/L NaCl, 5.6 mmol/L KCl, 1.2 mmol/L MgCl₂, 2.6 mmol/LCaCl₂, 10 mmol/L HEPES, and 4 mmol/L glucose (pH 7.4). After loading,the cells were rinsed and transferred to the imaging setup. The cellswere treated with individual drugs for 10 s, whereas fluorescence waselicited with the excitation wavelength alternating between 350 and 380nm, using a Polychromator V (TILL Photonics GmbH, Grafelfing, Germany)to provide illumination via a Zeiss Axiovert 100 microscope with a ZeissFluar 40

oil objective (Carl Zeiss, Jena, Germany). Images were captured using aCascade 512B CCD camera (Photometrics, Tucson, Ariz.) controlled withMetaFluor software (Molecular Devices, Sunnyvale, Calif.) at 0.5 Hzacquisition frequency. Ratios of the images obtained at 350 and 380 nmexcitation were used to show changes in the cytoplasmic calciumconcentration, according to the principles developed by Grynkiewicz etal. (31).

Drug Treatment of Nude Mice

Four- to 6-week-old male athymic nu/nu mice were obtained from Harlan(Indianapolis, Ind.) and implanted s.c. with 5×10⁵ U87 glioblastomacells as described in detail elsewhere (32). For the determination oftumor growth during continuous drug treatment for several weeks, DMC orrofecoxib was mixed with the daily chow (150 mg/kg for DMC; 40 mg/kg forrofecoxib), and tumor growth was monitored and recorded as described(32). For the analysis of short-term effects of drugs on CHOP expressionand tumor cell death in vivo, as well as for the determination of drugconcentrations in plasma and tumor tissue, tumor bearing animals weretreated with 30, 90, 150, or 180 mg/kg of drug per day for 50 h; eachanimal received one half of the daily dose of the respective drug every12 h via direct administration into the stomach with a stainless steelballhead feeding needle (Popper and Sons, Inc., New Hyde Park, N.Y.).All animals were sacrificed 2 h after the final application of drug, andtumors and blood were collected for analysis. In all experiments, theanimals were closely monitored with regard to body weight, foodconsumption, and clinical signs of toxicity; no differences betweennondrug-treated control animals and drug-treated animals were detected.

Extraction of Plasma for Liquid Chromatography Mass SpectrometryAnalysis

Blood was collected in heparinized syringes using cardiac puncture ofnude mice. The blood was allowed to settle at room temperature for 30min followed by centrifugation at 2,000 rpm for 5 min at 4° C. Theplasma was separated from the cells and transferred to a fresh tube. Toestablish a standard reference, 25 μL of 1.0 μg/mL DMC or celecoxib wereadded to 50 μL plasma from untreated control animals. For the testsamples, the same amount of DMC was added as an internal standard toplasma from animals that had been treated with celecoxib, whereas thesame amount of celecoxib was added as an internal standard to plasmafrom animals that had been treated with DMC. After thorough vortexing,plasma proteins were precipitated using 425 μL acetonitrile andvortexing for 1 min. The entire mixture was centrifuged at 4,500 rpm for5 min to separate the protein precipitate, and 400 μL of the supernatantwere transferred to a fresh tube. The sample was evaporated using asteady stream of air, and the dried residue was reconstituted using 150μL of mobile phase consisting of 80:20 (v/v) methanol:10 mmol/L ammoniumacetate (pH 4.5). To remove any undissolved precipitates, the sampleswere again centrifuged at 4,500 rpm for 5 min, and the supernatant wastransferred to a fresh tube. Ten microliters of each sample wereanalyzed in duplicate by liquid chromatography mass spectrometry.

To determine the amount of drug in each sample, an Agilent 1100high-pressure liquid chromatography system (Agilent Technologies, SantaClara, Calif.) coupled onto a Sciex API 3000 triple quadruple tandemmass spectrometer (Applied Biosystems, Foster City, Calif.) was used. Toseparate the analytes, a Thermo HyPURITY C18 column (50×4.6 nm, 3micron; Thermo Fisher Scientific, Inc., Waltham, Mass.) was used. Themobile phase consisted of 80:20 (v/v) methanol:10 mmol/L ammoniumacetate (pH 4.5). The flow rate to separate the analytes was 350 μL/min,where the retention time for DMC and celecoxib were 3.50 and 3.10 min,respectively. The analytes were then introduced into the Sciex API 3000,which was set in the negative ion mode. The level of DMC and celecoxibused the transition ions 394.0→330.2 and 380.0→316.2, respectively. Thelower level of quantification of this assay was established at 5 ng/nl.

Results

DMC is a close structural analogue of celecoxib that lacks the abilityto inhibit COX-2. To investigate whether this compound would be able toinduce the ESR, we treated various tumor cell lines with DMC, or inparallel with celecoxib, and determined the expression level of CHOPprotein. CHOP is a proapoptotic component of the ESR and is criticallyinvolved in the initiation of cell death after ER stress; therefore, weused it as a well-established indicator of ESR in our experimentalsystem. As shown in FIG. 3, both DMC and celecoxib were able to potentlyinduce CHOP in glioblastoma, breast carcinoma, pancreatic carcinoma,Burkitt's lymphoma, and multiple myeloma cell lines. Thus, both drugsseemed to stimulate the ESR, although celecoxib seemed to be somewhatless potent than DMC in glioblastoma and Burkitt's lymphoma cell lines.

To evaluate the extent of the ESR after DMC treatment, we analyzedadditional indicators of the ESR and compared the effects with thoseobtained with the use of thapsigargin, an inhibitor of sarcoplasmic/ERCa²⁺-ATPases that is frequently used as a strong model inducer of theESR. Cells were treated with either DMC or thapsigargin for varioustimes in parallel, and the expression levels of CHOP, GRP78, andcaspase-4, an ESR-specific caspase, were analyzed. FIG. 4 shows that DMCand thapsigargin stimulated the three selected ESR indicators in asimilar fashion. Both CHOP and GRP78 were substantially elevated, with aprominent increase noted at the 6-h time point and thereafter.Activation of caspase-4, which was indicated by the appearance of thecleaved (activated) form of this enzyme, was first noted at ˜24 h ofdrug treatment and persisted until the later (36 h) time point. Thus,the stimulation of the ESR was remarkably similar between DMC and themodel inducer thapsigargin, suggesting that the effects of DMC werequite potent in this context.

A prominent feature of the ESR is a general, transient down-regulationof overall protein synthesis, in combination with selectively increasedtranslation of ER stress proteins, such as GRP78 (33, 34). We thereforeinvestigated whether DMC and celecoxib would impair cellular translationby determining the incorporation of 35S-methionine into newly translatedproteins. As shown in FIG. 5, both drugs severely diminished the rate oftranslation in a concentration-dependent manner, with DMC beingnoticeably more potent. At 2 h of treatment, 60 μmol/L DMC and 80 μmol/Lcelecoxib were as effective as thapsigargin, and nearly as effective asthe potent translational inhibitor cycloheximide, and reduced ongoingtranslation by ˜90%. This inhibitory effect was transient, as cellsreturned to unrestricted, fully active protein synthesis by 18 h,despite the continuous presence of DMC or celecoxib (FIG. 5; data notshown for celecoxib). In addition, greatly increased translation ofGRP78 could be detected in DMC- and celecoxib-treated cells (FIG. 5;data not shown for celecoxib). Taken together, these results show thatDMC and celecoxib cause the typical features of ESR in drug-treatedcells.

Due to the striking similarities between the effects of DMC/celecoxiband those of thapsigargin, which is known to leak calcium from the ERand generate a calcium spike in the cytoplasm, we next determinedwhether DMC, and several coxibs and NSAIDs in comparison, would inducesuch a response as well. For this purpose, cells were loaded withFura-2/AM, exposed to 100 μmol/L of each drug, and the increase incytoplasmic calcium levels was measured. As shown in FIG. 6, DMC andcelecoxib caused a pronounced calcium spike, which could be observed ineach and every cell tested. In contrast, none of the other coxibs(rofecoxib and valdecoxib) or traditional NSAIDs (flurbiprofen,indomethacin, and sulindac) were able to elicit an elevation ofcytoplasmic calcium levels. Thus, DMC and celecoxib seemed to beuniquely able to mimic this aspect of thapsigargin, and the potentelevation of intracytoplasmic calcium levels by these drugs is entirelyconsistent with the generation of ESR, as documented in FIGS. 3-5 above.The average maximum calcium peak (FIG. 6B) caused by DMC was somewhatgreater than what was measured for celecoxib, but this difference wasnot statistically significant; however, overall calcium release (FIG.6A; area under the curve) was consistently 30% to 50% larger with DMC.

To further substantiate the uniqueness of DMC and celecoxib comparedwith other coxibs and NSAIDs, we next investigated how the observedeffects of these two drugs would compare with those of other coxibs andtraditional NSAIDs. First, we treated cells with DMC and the threecoxibs celecoxib, rofecoxib, and valdecoxib and determined theexpression levels of the ER stress indicator protein CHOP. At 50 μmol/L,DMC generated a pronounced induction of CHOP, which was detectable asearly as 4 h after the onset of drug treatment and continued to increaseup to 24 h (FIG. 6A). Treatment of cells with 50 μmol/L celecoxibresulted in a similar kinetic of CHOP induction, although the overalllevels were noticeable lower compared with DMC. In contrast, neitherrofecoxib nor valdecoxib at the same concentration resulted in anydetectable CHOP expression (FIG. 7A).

To investigate whether ESR induction could possibly be achieved athigher concentrations of rofecoxib and valdecoxib, the cells weretreated with further increased concentrations of each drug. However, asshown in FIG. 7B, even at concentrations of 75 or 100 μmol/L, neitherrofecoxib nor valdecoxib was able to stimulate any detectable increasein either CHOP or GRP78 protein. In comparison, DMC and celecoxibpotently induced the expression of both of these proteins, and onceagain, DMC.

We next investigated the relationship of ER stress induction and celldeath. FIG. 8A shows that 30 and 50 μmol/L DMC potently stimulated theESR, as indicated by the pronounced induction of CHOP, GRP78, andcaspase-4 cleavage/activation. In parallel, three variables of cellgrowth and cell death were investigated. First, a colony-forming assaywas done; this is an indicator of longterm survival that reveals thepercentage of individual cells that are able to survive and spawn acolony of new cell growth. Second, the traditional3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay wasused to determine short-term growth and survival, indicated primarily bythe metabolic activity of the entire cell population. Third, terminaldeoxynucleotidyl transferase-mediated dUTP nick end labeling assay wasdone to quantify the fraction of cells undergoing apoptosis. As shown inFIG. 6A, the induction of ESR by DMC closely correlated with greatlyreduced survival in colony-forming assays, with reduced cellularactivity in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideassays, and with substantially increased apoptosis.

Celecoxib as well was able to induce cell death/apoptosis (FIG. 5B) andreduced the viability of the cell culture (FIG. 8C), although itspotency was clearly less than that of DMC (as indicated by therequirement of higher concentrations). However, none of the other coxibs(rofecoxib and valdecoxib) or traditional NSAIDs (flurbiprofen,indomethacin, and sulindac) had any detectable effect on cell growth andsurvival and did not induce apoptosis even at concentrations of up to100 μmol/L (FIGS. 8B and C). Thus, taken together, these resultsindicate that celecoxib is unique among these coxibs/NSAIDs because ofits superior potency to stimulate the ESR and initiate tumor cell death;in addition, its derivative DMC seems even more effective, clearlyarguing that the inhibition of COX-2 is not required to achieve theseeffects.

We next investigated the contribution of the ESR to reduced tumor cellgrowth in response to treatment with DMC or celecoxib. For this purpose,we applied specific siRNAs to knock down the expression of either GRP78(representing the protective branch of the ESR) or caspase-4(representing the proapoptotic branch). Glioblastoma cells weretransfected with these siRNAs and treated with drug for 48 h, and thepercentage of surviving cells was determined with the use of the colonyformation assay (FIG. 9A). As a control, a si-GFP was included in theseexperiments; furthermore, the efficiency of target knockdown by eachspecific siRNA was confirmed by Western blot analysis of caspase-4 andGRP78 protein (FIG. 9B). For GRP78-siRNA, the cells became moresensitive and there was less cell survival when GRP78 levels werereduced. For caspase-siRNA, the opposite was observed: we found that thesensitivity of cells to drug treatment was significantly reduced (i.e.,cell survival after treatment with celecoxib or DMC was increased whencaspase-4 expression was diminished by siRNA). Thus, these results agreewith the current model of ESR, where GRP78 represents the protectivearm, whereas caspase-4 is proapoptotic and necessary for the executionof cell death after ER stress; our results indicate that DMC andcelecoxib overpower the protective effort by GRP78 and induce cell deathvia the stimulation of caspase-4 activity.

In an effort to determine whether ER stress might be relevant during thein vivo antitumor activity of DMC, we used a xenograft nude mouse tumormodel and investigated the expression of CHOP protein in tumor tissuefrom animals treated with DMC or rofecoxib. As shown in FIG. 10, CHOPprotein was barely detectable in tumor tissue from control animals(i.e., in the absence of any drug treatment). In contrast, when animalswere fed with DMC for 50 h, there was a large increase in CHOP proteinexpression in their tumor tissue. In comparison, when animals receivedrofecoxib, no such increase was observed (FIG. 10). In addition, thehighly elevated amount of CHOP protein after DMC treatment correlatedwith significantly increased apoptosis in the tumor tissue, whereastumors from rofecoxib-treated animals did not display elevated levels ofapoptosis (FIG. 10). Furthermore, on longer-term therapy oftumor-bearing animals with either DMC or rofecoxib, it became apparentthat only DMC caused significantly reduced tumor growth (FIG. 11),indicating that the induction of ESR and apoptosis by DMC indeedtranslated into overall reduced tumor growth in this xenograft model.

Finally, as an extension of earlier pharmacokinetic/pharmacodynamicdeterminations of DMC and celecoxib in nude mice (20), we measured theconcentration of drug (DMC and celecoxib) in blood and tumor tissuesfrom our experimental animals. Tumor-bearing animals were treated for 2days with daily dosages ranging from 30 to 180 mg/kg DMC or celecoxib,and the absolute levels (C_(max)) of each drug were determined by liquidchromatography mass spectrometry. As presented in the Table 2, maximaldrug concentrations in plasma and tumor tissue increased as dailydosages increased and reached peak levels of 45 μmol/L in the plasmafrom animals treated with the highest dose of 180 mg/kg. Intriguingly,however, the concentrations in tumor tissues were two orders ofmagnitude lower than the corresponding plasma concentrations; in animalsreceiving the highest daily dose of 180 mg/kg, tumor tissueconcentrations approximately equivalent to only 0.25 μmol/L werereached. Nonetheless, in all of these tumor tissues, increased levels ofCHOP expression were observed, whereas tumor tissues from non-drugtreated or rofecoxib treated (30-180 mg/kg) animals were consistentlynegative for this ER stress indicator protein (see FIG. 10). In general,tumor tissues from animals treated with the lowest dosages of DMC orcelecoxib were positive for CHOP, although tumors from those animalsexposed to much higher concentrations stained more intensely for thisprotein. Importantly, these results show that despite the hugedifference between the drug concentrations used in vitro and thosemeasured in vivo, in both cases, ER stress and subsequent tumor celldeath were achieved.

Example 2 Aggravated Endoplasmic Reticulum Stress as a Basis forEnhanced Glioblastoma Cell Killing by Bortezomib in Combination withCelecoxib or its Non-Coxib Analogue, 2,5-Dimethyl-Celecoxib Materialsand Methods Materials

Bortezomib was obtained from the pharmacy as 3.5 mg Velcade suspended in3.5 mL saline (Millennium Pharmaceuticals). Celecoxib was obtained ascapsules from the pharmacy or synthesized in our laboratory according topreviously published procedures (24). DMC is a close structural analogueof celecoxib, where the 5-aryl moiety has been altered by replacing4-methylphenyl with 2,5-dimethylphenyl; this compound was synthesized inour laboratory according to previously published procedures (19).Celecoxib and DMC were dissolved in DMSO at 100 mmol/L (stock solution)and added to the cell culture medium in a manner that kept the finalconcentration of solvent below 0.1%. The COX-2-inhibitory activity offreshly synthesized celecoxib, and lack thereof in DMC, was confirmed invitro with the use of purified COX-2 protein (see, for example, ref.42).

Cell Lines and Culture Conditions

All cells were propagated in DMEM (Cell gro) supplemented with 10% fetalbovine serum, 100 units/mL penicillin, and 0.1 mg/mL streptomycin in ahumidified incubator at 37° C. and a 5% CO₂ atmosphere. Four humanglioblastoma cell lines (LN229, U251, T98G, and U87MG) and one multiplemyeloma cell line (RPMI/8226) were used. T98G, U87MG, and RPMI/8226cells were obtained from the American Type Culture Collection. LN229 andU251 were obtained from Frank B. Furnari (Ludwig Institute of CancerResearch, La Jolla, Calif.). COX-2 expression levels and drug effects onprostaglandin production in these cells have been published elsewhere.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Assay

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assays were performed in 96-well plates with the use of 3.0×10³ to8.0×10³ cells per well as described in detail elsewhere (19).

Cell Death ELISA

Cells were plated in 96-well plates in quadruplicates at 1,000 cells/mL(100 μL/well). The next day, they were treated with drugs for 24 h andanalyzed for the presence of histone-complexed DNA fragments with theuse of a commercially available ELISA kit (Roche Diagnostics) accordingto the manufacturer's instructions. The kit was used in a manner as tospecifically quantitate apoptosis rather than necrosis.

Immunoblots and Immunohistochemical Staining

Total cell lysates were prepared and analyzed by Western blot analysisas described earlier (19). Immunohistochemical analysis of proteinexpression in tumor tissues was performed with the use of the Vectastainavidin-biotin complex method kit (Vector Laboratories) as describedpreviously (32). The primary antibodies were purchased from CellSignaling Technology or Santa Cruz Biotechnology, Inc. and usedaccording to the manufacturers' recommendations. All immunoblots andstainings were repeated at least once to confirm the results.

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End LabelingStaining of Tumor Tissue

Apoptosis in tumor sections was measured quantitatively with the use ofthe terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling (TUNEL) assay. All components for this procedure were from theApopTag In Situ Apoptosis Detection kit (Chemicon), which was usedaccording to the manufacturer's instructions. The percentage ofTUNEL-positive cells for each tumor section was determined from 10random photomicrographs taken at 200 magnification.

Transfections and Colony Formation Assay

The different small interfering RNAs (siRNA) were synthesized at themicrochemical core laboratory of the University of SouthernCalifornia/Norris Comprehensive Cancer Center; their sequences aredescribed in ref. 32. Transfections of cells with these siRNAs andsubsequent analysis of cell survival by colony formation assay have beendescribed in detail elsewhere (32).

Drug Treatment of Nude Mice

Four- to 6-week-old male athymic nu/nu mice were obtained from Harlanand implanted s.c. with 5×10⁵ U87 glioblastoma cells. Once tumors of˜300 mm³ had developed, the animals received drug treatments. Velcadewas given as a single dose via tail vein injection. DMC was given twicedaily (one half of the daily dose every 12 h) via direct administrationinto the stomach with a stainless steel ball-head feeding needle (Popperand Sons, Inc.). After a total of 50 h, the animals were sacrificed andtumors were collected for analysis. In all experiments, the animals wereclosely monitored with regards to body weight, food consumption, andclinical signs of toxicity; no differences between non-drug-treatedcontrol animals and drug-treated animals were detected.

Results

Glioblastoma multiforme represents a particularly difficult-to-treattype of cancer with dismal prognosis. Because more effective therapiesare urgently needed, we chose various human glioblastoma cell lines as amodel to investigate combination effects of our selected drugs. Toestablish the concentration of each drug that would result in 50%inhibition of cell growth (IC₅₀), we first treated each cell lineindividually with either bortezomib, celecoxib, or the non-coxibcelecoxib analogue DMC. The resulting ICSO after 48 h of treatment withbortezomib was ˜10 nmol/L for the U251, U87MG, and T98G glioblastomacell lines and slightly below 5 nmol/L for the LN229 glioblastoma cellline (FIG. 12A). Because bortezomib was developed for multiple myelomatherapy and is highly cytotoxic in such tumor cell lines, we alsodetermined its IC₅₀ in a representative multiple myeloma cell line,RPMI/8226, for comparison purposes. As shown in FIG. 12A, and asexpected, RPMI/8226 cells exhibited high sensitivity toward bortezomib;however, this cell type was not more sensitive than the glioblastomacell lines. This finding established that glioblastoma cells wereexquisitely sensitive to bortezomib, which supported our rationale forinvestigating this drug as a potential glioblastoma therapy.

We also established the IC₅₀ for celecoxib and DMC in all fourglioblastoma cell lines (FIGS. 12A and B). Celecoxib displayed an IC₅₀of ˜50 μmol/L in U251, U87MG, and T98G cells, whereas DMC was somewhatmore potent with an IC₅₀ of slightly below 40 μmol/L. The LN229 cellline (which exhibited the greatest sensitivity to bortezomib; FIG. 12A)was overall slightly less sensitive to either celecoxib or DMC (FIGS.12A and B).

To determine the ability of bortezomib, celecoxib, and DMC to induce theESR in glioblastoma cells, we treated U251 cells with increasingconcentrations of each drug increased expression of GRP78 and CHOP,indicating that the ESR was triggered. In addition, this drug alsostimulated the activation of the ER stress-associated procaspase-4, asindicated by the appearance of the cleaved (i.e., activated) form ofthis enzyme. To verify that bortezomib, at the concentrations we used,exerted its established function (i.e., inhibition of the proteasome),we also investigated the accumulation of polyubiquitinated proteins. Asshown in FIG. 13A, the induction of ER stress markers coincided with theappearance of highly elevated levels of polyubiquitinated proteins,indicating that inhibition of the proteasome correlated with theinduction of ER stress.

The same targets were also investigated after treatment of cells witheither celecoxib or DMC. In all our experiments, celecoxib and DMCconsistently generated the same outcome, except that DMC was slightlymore potent [for this reason, and also because the induction of ERstress by celecoxib has been reported earlier, we will focus primarilyon the results obtained with DMC]. As shown in FIG. 3B, DMC treatmentresulted in strong induction of GRP78 and CHOP and activation ofcaspase-4, indicating that this drug triggered ER stress. However, thisdrug did not cause substantial accumulation of polyubiquitinatedproteins, consistent with the expectation that its mechanism of actionwas different from that of bortezomib.

We next examined the effects of combination drug treatments onglioblastoma cell growth and survival. For this purpose, we combinedbortezomib with either celecoxib or DMC at concentrations thatrepresented approximate IC₅₀ values so that potentially enhancingeffects would become apparent. FIG. 14A depicts a visual display of cellnumber and morphology, whereas FIG. 14B presents the quantified outcome.FIG. 14A reveals that individual drug treatment resulted in a smallerincrease in cell number and the presence of fewer mitotic figures; incontrast, combination drug treatment resulted in noticeable cell lossand apparent cell death. These visual impressions were complemented bycounting the number of viable cells over the course of drug treatment(at 8, 24, and 48 h). As displayed in FIG. 14B, single-drug treatmentallowed initial cell proliferation (i.e., increase in cell numbersbetween 8 and 24 h) and then exerted presumed cytostatic effects (i.e.,the overall number of cells did not change between 24 and 48 h). Incontrast, when the drugs were applied in combination, there was noincrease in cell numbers at the 24-h time point and substantial loss ofviable cells between 24 and 48 h, indicating potent cytotoxic effects.

The extent of drug-induced cell death and survival was furtherinvestigated by cell death ELISA, which quantitates the amount ofapoptosis in the entire culture, and by colony formation assays, whichdetermine the number of individual cells able to survive drug treatmentlong-term and spawn a colony of clonal descendants. FIG. 14C shows thattreatment of glioblastoma cells with individual drugs caused a smallincrease in apoptotic cell death, whereas the combined treatment withbortezomib and celecoxib or DMC resulted in greatly increased celldeath. In colony formation assays, 5 mmol/L bortezomib reduced thenumber of emerging colonies by 50% (FIG. 14D). The chosen concentrationsof celecoxib and DMC by themselves exerted only minor inhibitory effects(reduction of colony number by approximately 10-15%). In contrast, whencells were treated with bortezomib and celecoxib or DMC in combination,colony survival was greatly reduced by >90% and 97%, respectively.

Each assay used in FIG. 14 was applied to several different glioblastomacell lines and varying concentrations of drug combinations were used(data not shown). In all cases, very similar outcomes were achieved,clearly indicating that the combination of these drugs results ingreatly increased cytotoxicity and substantially reduced cell survivalcompared with treatment with each drug individually. Furthermore, wecalculated the combination index (CI) from conventional MTT assays whereincreasing concentrations of each drug were combined (data not shown)and obtained a CI<1, revealing that the drug combination effects weresynergistic.

We next investigated the potential contribution of the ESR to theabove-presented combination drug effects. U87MG and T98G cells weretreated with the same drug combinations as above, and various componentsof the ESR system and cell death machinery were analyzed. As shown inFIG. 15, individual drug treatments resulted in increased expression ofGRP78, and combination drug treatments increased expression of thisprotein further. Levels of the proapoptotic CHOP protein were weaklyincreased by single-drug treatments but were more strongly elevated bycombination treatments. The activity of c-Jun NH2-terminal kinase (JNK),a critical proapoptotic component of the ESR, was investigated withantibodies specifically recognizing the phosphorylated (i.e., active)form of this kinase. We found that combination drug treatments, but notindividual drug treatments, resulted in greatly increased JNK activity(FIG. 15A). Taken together, these results indicate that bortezomib, whencombined with either celecoxib or DMC, caused stronger ESR inductionthan either drug alone. Similar results were also obtained with the useof LN229 and U251 cells (data not shown).

To establish whether drug-induced ER stress and cellular apoptosis wereonly correlative or were causally related, we specifically reduced theexpression of the ESR component GRP78. If drug-induced cell death wascontrolled by the ESR, we would expect that reduced levels of GRP78,which functions as a major protective component of the ESR, would leadto further increased cell death. U251 cells were transfected with siRNAagainst GRP78; as a control, cells were transfected with siRNA against atarget not present in mammalian cells [i.e., green fluorescent protein(GFP)]. Both siGRP78-transfected and siGFP-transfected cells weretreated with bortezomib together with celecoxib or DMC, and the numberof surviving cells was determined by colony formation assay. As shown inFIG. 16A, cell survival was significantly (P<0.002) decreased in cellsharboring reduced levels of GRP78; cell survival after drug treatmentwas 63% to 68% in cells transfected with siGFP but was decreased to 40%to 42% in cells transfected with siGRP78. Down-regulation of GRP78expression by siGRP78 was confirmed by Western blot analysis; however,whereas the presence of siGRP78 reduced basal levels of GRP78 below thedetection limit, the siRNA could not completely block the induction ofGRP78 by drug treatment, as lower levels of induced GRP78 protein couldstill be detected (FIG. 16B). Nonetheless, under all conditions, theoverall amount of GRP78 protein was lower in siGRP78-transfected cellsthan in siGFP-transfected cells. Concurrently, siGRP78-transfected cellsexhibited significantly increased chemosensitivity, indicating that theESR played a causal role in triggering cell death induced by bortezomibin combination with celecoxib or DMC.

Finally, we determined whether the above-described in vitro events wouldalso take place in vivo. U87MG cells were implanted s.c. into nude mice,and after sizable tumors had formed, the animals remained untreated orwere treated with drugs. Celecoxib was not included in this experimentbecause previous studies had already shown that this drug potentlystimulated the ESR in vivo (39, 40); furthermore, all known experimentsthat compared DMC to celecoxib have shown that both drugs reliablyachieve the same tumor-suppressive outcome in vitro and in vivo, exceptthat DMC consistently displays somewhat greater apoptosis-inducingpotency. Therefore, we decided on DMC as the more potent drug of choicefor the in vivo combination experiments with bortezomib.

We had shown previously that DMC by itself quite potently triggers ERstress in tumor tissue in vivo (39), and this effect begins to appear atdosages of >10 mg/kg. Considering these earlier results, we chose 7.5mg/kg as a potentially useful dosage for this combination experiment; wereasoned that suboptimal dosages of DMC would allow the combinationeffects to emerge. Tumor bearing animals were treated with DMC orbortezomib alone or in combination for 2 days (a relatively shorttreatment period was chosen because we wanted to focus on the earlymechanisms that initiate cell death rather than the later conditionsthat dominate when the tumor cells are dying or already are dead). Tumortissue was analyzed for the expression of CHOP (as a marker of ERstress) and stained by TUNEL (to visualize the extent of apoptotic celldeath).

We found that DMC, at this very low dosage, did not cause much elevatedCHOP expression nor did it substantially increase TUNEL staining (FIG.17A). Bortezomib treatment by itself resulted in a larger fraction ofCHOP-positive cells, concomitant with increased TUNEL staining. Incomparison, when bortezomib was given together with DMC, there was avery strong induction of CHOP, which could be detected in every singlecell, and an even greater increase in the number of TUNEL-positive cells(FIG. 17A). When the number of TUNEL-positive cells in the varioustreatment groups was quantitated, it became apparent that combinationtreatment resulted in significantly more cell death than individual drugtreatment (FIG. 17B); that is, cell death in response to treatment withbortezomib plus DMC was 4.9- and 3.0-fold higher than in animals treatedwith DMC or bortezomib alone, respectively. Thus, combination drugtreatment resulted in substantially higher levels of ER stress and agreater amount of cell death than individual drug treatments andestablished that aggravated ER stress and enhanced glioblastoma cellkilling could be achieved in vivo as well.

Example 3 Aggravated Endoplasmic Reticulum Stress as a Basis forEnhanced Cell Killing of Tumor-Associated Brain Endothelial Cells byTemozolomide in Combination with the Non-Coxib Celecoxib Analogue,2,5-Dimethyl-Celecoxib (DMC) Materials and Methods

Tumor-associated Brain Endothelial Cells (TuBEC) were treated with TMZ(300 uM) and 20 uM DMC for 4.8 hours. The drugs were removed and cellswere incubated for another 12 days; at that time cytotoxicity wasevaluated using the trypan blue exclusion technique. Results arepresented as number of viable cells per group.

Results

Temozolomide (TMZ), the standard of treatment for gliomas, is veryeffective against the tumor cells. However, this drug causes littlecytotoxicity on the tumor-associated brain endothelial cells (TuBEC).Our experiments, summarized in FIG. 18 have shown that the ERaggravating agent DMC increased the susceptibility of the tumor vascularendothelial cells susceptible to TMZ. Thus DMC functions as ananti-angiogenic agent by chemosensitizing these cells to knowntherapeutic drugs.

Although the present invention has been described in terms of specificexemplary embodiments and examples, it will be appreciated that theembodiments disclosed herein are for illustrative purposes only andvarious modifications and alterations might be made by those skilled inthe art without departing from the spirit and scope of the invention asset forth in the appended claims.

TABLE 1 Apoptosis-triggering Strategies Strategy Target Approach Stageof development Proapoptotic approaches Introduction of proapoptoticTRAIL Recombinant protein Clinical trials planned players Apoptin Genetherapy Preclinical Caspases Gene therapy Preclinical Modulation ofantiapoptotic Mitochondria: genes or pathways Proapoptotic molecules(Bax, BCL-Xs) Gene therapy Preclinical Downregulate antiapoptoticmolecules ODNs Phase II/III (Bcl-2, Bcl-XL) Direct effect onmitochondria Lonidamine, arsenite Phase III Direct effect on pores PK11195 Preclinical Restoration or manipulation p53 Gene therapy PhaseII/III of tumor suppressor genes Retinoblastoma Gene therapy PreclinicalFHIT Gene therapy Clinical trials planned Permissive approachesOncogenes PI3k LY294002 Preclinical Ras Small molecules, ODNs PhaseII/III BCR-ABL Small molecule (STI-571) Phase III NFκB ODNs Phase I/IIProtensome inhibitors PS-341 Phase II c-raf ODNs Phase II c-myb ODNsPreclinical Cell cycle modulators UCN-01, flavopiridol Phase III

TABLE 2 Drug concentrations in plasma and tumor tissue Drug and Maximumplasma Tumor tissue Animal dosage, levels*, levels*, ng/mg no. mg/kg/dμg/L (μmol/L) (approximately μmol/L) 1 No drug <0.5^(†) <0.2^(‡)treatment 2 Cxb 30 6,950 (18.2) 17.53 (0.046) 3 Cxb 90 9,450 (24.8)44.03 (0.116) 4 Cxb 150 14,000 (36.8) 61.23 (0.161) 5 Cxb 180 17,000(44.6) 94.78 (0.249) 6 DMC 30 3,035 (7.6) 8.27 (0.021) 7 DMC 90 3,190(8.0) 14.44 (0.037) 8 DMC 150 9,500 (23.8) 39.04 (0.099) 9 DMC 18018,200 (45.5) 110.42 (0.280) Abbreviation: Cxb, celecoxib. *Average oftwo measurements. ^(†)Detection limit in blood was ~5 μg/L ^(‡)Detectionlimit in tumor tissue was ~5 ng/mg.

REFERENCES

-   1. Kerr, J. F., Wyllie, A. H., and Currie, A. R. Apoptosis: a basic    biological phenomenon with wide-ranging implications in tissue    kinetics. Br. J, Cancer, 26: 239-257, 1972.-   2. Hengartner M. O. The biochemistry of apoptosis. Nature, 407:    770-776, 2000.-   3. Thompson C. B. Apoptosis in the pathogenesis and treatment of    disease. Science, 267: 1456-1462, 1995.-   4. Ferreira G. C., et al, Clinical Cancer Research Vol. 8,    2024-2034, July 2002.-   5. Boyce M, Yuan J. Cellular response to endoplasmic reticulum    stress: a matter of life or death. Cell Death Differ 2006;    13:363-73.-   6. Wu J, Kaufman R J. From acute ER stress to physiological roles of    the unfolded protein Response. Cell Death Differ 2006; 13:374-84.-   7. Ulrich C M, Bigler J, Potter J D. Non-steroidal anti-inflammatory    drugs for cancer prevention: promise, perils, and pharmacogenetics.    Nat Rev Cancer 2006; 6:130-40.-   8. Hitomi J, Katayama T, Eguchi Y, et al. Involvement of caspase-4    in endoplasmic reticulum stress-induced apoptosis and Ah-induced    cell death. J Cell Biol 2004; 165:347-56.-   9. Oyadomari S, Mori M. Roles of CHOP/GADD153 in endoplasmic    reticulum stress. Cell Death Differ 2004; 11:381-9.-   10. Parente L, Perretti M. Advances in the pathophysiology of    constitutive and inducible cyclooxygenases: two enzymes in the    spotlight. Biochem Pharmacol 2003; 65:153-9.-   11. Yoshida H, Okada T, Haze K, Yanagi H, Yura T, Negishi M and Mori    K (2001) Endoplasmic reticulum stress-induced formation of    transcription factor complex ERSF including NF-Y (CBF) and    activating transcription factors 6alpha and 6beta that activates the    mammalian unfolded protein response. Mol. Cell. Biol. 21: 1239-1248.-   12. Yoshida H, Matsui T, Yamamoto A, Okada T and Mori K (2001) XBP1    mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress    to produce a highly active transcription factor. Cell 107: 881-891.-   13. Calfon M, Zeng H, Urano F, Till J H, Hubbard S R, Harding H P,    Clark S G and Ron D (2002) IRE1 couples endoplasmic reticulum load    to secretory capacity by processing the XBP-1 mRNA. Nature 415:    92-96-   14. Lee K, Tirasophon W, Shen X, Michalak M, Prywes R, Okada T,    Yoshida H, Mori K and Kaufman R J (2002) IRE1-mediated    unconventional mRNA splicing and S2P-mediated ATF6 cleavage merge to    regulate XBP1 in signaling the unfolded protein response. Genes Dev.    16: 452-466-   15. Wang Y, Shen J, Arenzana N, Tirasophon W, Kaufman R J and Prywes    R (2000) Activation of ATF6 and an ATF6 DNA binding site by the    endoplasmic reticulum stress response. J. Biol. Chem. 275:    27013-27020-   16. Ubeda M and Habener J F (2000) CHOP gene expression in response    to endoplasmic-reticular stress requires NFY interaction with    different domains of a conserved DNA-binding element. Nucleic Acids    Res. 28: 4987-4997-   17. Arico S, Pattingre S, Bauvy C, et al. Celecoxib induces    apoptosis by inhibiting 3-phosphoinositide-dependent protein    kinase-1 activity in the human colon cancer HT-29 cell line. J Biol    Chem 2002; 277: 27613-21.-   18. Hanif R, Pittas A, Feng Y, et al. Effects of nonsteroidal    anti-inflammatory drugs on proliferation and on induction of    apoptosis in colon cancer cells by a prostaglandin-independent    pathway. Biochem Pharmacol 1996; 52:237-45.-   19. Kardosh A, Blumenthal M, Wang W J, et al. Differential effects    of selective COX-2 inhibitors on cell cycle regulation and    proliferation of glioblastoma cell lines. Cancer Biol Ther 2004;    3:9-16.-   20. Kulp S K, Yang Y T, Hung C C, et al.    3-Phosphoinositide-dependent protein kinase-1/Akt signaling    represents a major cyclooxygenase-2-independent target for celecoxib    in prostate cancer cells. Cancer Res 2004; 64:1444-51.-   21. Shureiqi I, Chen D, Lotan R, et al. 15-Lipoxygenase-1 mediates    nonsteroidal anti-inflammatory drug-induced apoptosis independently    of cyclooxygenase-2 in colon cancer cells. Cancer Res 2000;    60:6846-50.-   22. Tegeder I, Pfeilschifter J, Geisslinger G.    Cyclooxygenase-independent actions of cyclooxygenase inhibitors.    FASSEB J 2001; 15:2057-72.-   23. Zhang X, Morham S G, Langenbach R, et al. Malignant    transformation and antineoplastic actions of nonsteroidal    antiinflammatory drugs (NSAIDs) on cyclooxygenase-null embryo    fibroblasts. J Exp Med 1999; 190:451-9.-   24. Penning T D, Talley J J, Bertenshaw S R, et al. Synthesis and    biological evaluation of the 1,5-diarylpyrazole class of    cyclooxygenase-2 inhibitors: identification of    4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide    (SC-58635, celecoxib). J Med Chem 1997; 40: 1347-65.-   25. Talley J J, Brown D L, Carter J S, et al.    4-[5-Methyl-3-phenylisoxazol-4-yl]-benzenesulfonamide, valdecoxib: a    potent and selective inhibitor of COX-2. J Med Chem 2000; 43:775-7.-   26. Chan C C, Boyce S, Brideau C, et al. Rofecoxib [Vioxx, MK-0966;    4-(4′-methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone]: a potent and    orally active cyclooxygenase-2 inhibitor. Pharmacological and    biochemical profiles. J Pharmacol Exp Ther 1999; 290:551-60.-   27. Prasit P, Wang Z, Brideau C, et al. The discovery of rofecoxib,    [MK 966, Vioxx,    4-(4′-methylsulfonylphenyl)-3-phenyl-2(5H)-furanone], an orally    active cyclooxygenase-2-inhibitor. Bioorg Med Chem Lett 1999; 9:    1773-8.-   28. Harlow E, Lane D. Using antibodies: a laboratory manual. Cold    Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 1999. p.    267-309.-   29. Wu R-C, Schonthal A H. Activation of p53-21waf1 pathway in    response to disruption of cell-matrix interactions. J Biol Chem    1997; 272:29091-8.-   30. Heatwole V M. TUNEL assay for apoptotic cells. Methods Mol Biol    1999; 115:141-8.-   31. Grynkiewicz G, Poenie M, Tsien R Y. A new generation of Ca²⁺    indicators with greatly improved fluorescence properties. J Biol    Chem 1985; 260:3440-50.-   32. Pyrko P, Soriano N, Kardosh A, et al. Downregulation of    surviving expression and concomitant induction of apoptosis by    celecoxib and its non-cyclooxygenase-2-inhibitory analog,    dimethyl-celecoxib (DMC), in tumor cells in vitro and in vivo. Mol    Cancer 2006; 5:19.-   33. Luo S, Baumeister P, Yang S, et al. Induction of Grp78/BiP by    translational block: activation of the Grp78 promoter by ATF4    through and upstream ATF/CRE site independent of the endoplasmic    reticulum stress elements. J Biol Chem 2003; 278:37375-85.-   34. Ma Y, Hendershot L M. The role of the unfolded protein response    in tumour development: friend or foe? Nat Rev Cancer 2004; 4:966-77.-   35. Kim S H, Hwang C I, Park W Y, et al. GADD153 mediates    celecoxibinduced apoptosis in cervical cancer cells. Carcinogenesis    2006; 28: 223-31.-   36. Trifan O C, Durham W F, Salazar V S, et al. Cyclooxygenase-2    inhibition with celecoxib enhances antitumor efficacy and reduces    diarrhea side effect of CPT-11. Cancer Res 2002; 62:5778-84.-   37. Roh J L, Sung M W, Park S W, et al. Celecoxib can prevent tumor    growth and distant metastasis in postoperative setting. Cancer Res    2004; 64:3230-5.-   38. Williams C S, Watson A J, Sheng H, et al. Celecoxib prevents    tumor growth in vivo without toxicity to normal gut: lack of    correlation between in vitro and in vivo models. Cancer Res 2000;    60:6045-51.-   39. Tegeder I, Pfeilschifter J, Geisslinger G.    Cyclooxygenase-independent actions of cyclooxygenase inhibitors.    FASEB J 2001; 15:2057-72.-   40. Backhus L M, Petasis N A, Uddin J, et al. Dimethyl-celecoxib as    a novel non-COX-2 therapy in the treatment of lung cancer. J Thorac    Cardiovasc Surg 2005; 130: 1406-12.-   41. Johnson A J, Song X, Hsu A, et al. Apoptosis signaling pathways    mediated by cyclooxygenase-2 inhibitors in prostate cancer cells.    Adv Enzyme Regul 2001; 41:221-35.-   42. Schonthal A H. Antitumor properties of dimethyl celecoxib, a    derivative of celecoxib that does not inhibit cyclooxygenase-2:    implications for glioblastoma therapy. Neurosurgical Focus 2006;    20:1-10.

1. A method of inducing or aggravating stress in a cell's endoplasmicreticulum (ER) to trigger apoptosis, comprising: inhibiting SERCAactivity selectively in said cell without inhibiting COX-2 activity; andelevating the expression of CHOP in said cell, wherein, the combinationof inhibiting SERCA activity and elevating CHOP expression results in acondition favorable for initiation of apoptosis in the cell.
 2. Themethod of claim 1, wherein the step of selectively inhibiting SERCA isaccomplished by administering a pharmaceutically effective amount of anER-stress aggravating agent capable of selectively inhibiting SERCA,directly or indirectly, for a duration sufficient to elevate cytoplasmiccalcium concentration significantly above normal level so as to causeapoptosis.
 3. The method of claim 2, wherein said ER-stress aggravatingagent is one that does not inhibit COX-2.
 4. The method of claim 2,wherein said ER-stress aggravating agent does not trigger release ofhistamine.
 5. The method of claim 3, wherein said ER-stress aggravatingagent comprises a compound having a general formula:

wherein, R₁ is methyl, fluoromethyl, difluoromethyl, trifluoromethyl,alkyl, fluoroalkyl, difluoroalkyl, trifluoroalkyl, polyfluoroalkyl,hydroxyalkyl, or carboxyalkyl; R₂ is hydrogen, fluoro, chloro, bromo;fluoromethyl, difluoromethyl, trifluoromethyl, alkyl, fluoroalkyl,difluoroalkyl, trifluoroalkyl, polyfluoroalkyl, hydroxyalkyl, orcarboxyalkyl; R₃-R₇ are independently selected from a group consistingof: hydrogen, fluoro, chloro, bromo, alloxy, alkyl, fluoroalkyl,difluoroalkyl, trifluoroalkyl, polyfluoroalkyl, hydroxyalkyl, orcarboxyalkyl, aryl and heteroaryl; R₈-R₁₁ are independently selectedfrom a group consisting of: hydrogen, fluoro, chloro, bromo;fluoromethyl, difluoromethyl, trifluoromethyl, alkyl, fluoroalkyl,difluoroalkyl, trifluoroalkyl, polyfluoroalkyl, hydroxyalkyl,carboxyalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, orheteroaryl; and R₁₂ is hydrogen, acetyl, acyl, alkyl, fluoroalkyl,difluoroalkyl, trifluoroalkyl, polyfluoroalkyl, hydroxyalkyl,carboxyalkyl; aminoacyl, aminoalkyl, cycloalkyl, heterocyclic, aryl, orheteroaryl.
 6. The method of claim 5, wherein R₁ is trifluoromethyl. 7.The method of claim 5, wherein R₂, R₈-R₁₂ are hydrogen.
 8. The method ofclaim 5, wherein R₄, R₅, and R₇ are hydrogen.
 9. The method of claim 5,wherein R₁ is trifluoromethyl, R₂, R₄, R₅, R₇-R₁₂ are hydrogen.
 10. Themethod of claim 5, wherein R₃ and R₆ are selected from a groupconsisting of: hydrogen, fluoro, chloro, bromo; fluoromethyl,difluoromethyl, trifluoromethyl, alkyl, aryl, heteroaryl, fluoroalkyl,difluoroalkyl, trifluoroalkyl, polyfluoroalkyl, hydroxyalkyl, orcarboxyalkyl.
 11. The method of claim 2, wherein said ER-stressaggravating agent is4-[5-(2,5-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide,4-[5-(2,5-di(trifluoromethyl)phenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamideand4-[5-(2,5-dibromophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamideor an analogue thereof.
 12. The method of claim 2, wherein saidER-stress aggravating agent comprises a prodrug that will metabolizeinto a SERCA inhibitor.
 13. The method of claim 1, further comprising astep of applying a second ER-stress aggravating agent capable ofincreasing the concentration of misfolded or damaged proteins in the ER.14. The method of claim 13, wherein said second ER-stress aggravatingagent comprises a proteasome inhibitor or a protease inhibitor.
 15. Themethod of claim 14, wherein said proteasome inhibitor is bortezomib oran analogue thereof.
 16. The method of claim 14, wherein said proteaseinhibitor is an HIV protease inhibitor selected from: nelfinavir,atazanavir, fosamprenavir, ritonavir, indinavir or an analogue thereof.17. The method of claim 1, further comprising a step of applying anapoptosis enhancer.
 18. The method of claim 17, wherein said apoptosisenhancer is one that is capable of upregulating the expression of CHOP,overcoming a protective function of GRP78, and activating a caspase. 19.The method of claim 17, wherein said apoptosis enhancer is a siRNA forGRP78 or an inhibitor of GRP78 function.
 20. A method for screening,selecting, or designing compounds useful for inducing or aggravating ERstress in a cell to trigger apoptosis in the cell, comprising: obtaininginformation about a test compound, wherein said information comprisesSERCA inhibition activity, COX-2 inhibition activity, and histaminerelease activity; and identifying said test compound as a potentialER-stress aggravating agent if said compound is an inhibitor of SERCA,and is not an inhibitor of COX-2.
 21. The method of claim 20, whereinsaid obtaining step comprises performing biochemical assays, cell-basedassays, or online database search.
 22. The method of claim 20, furthercomprising repeating the steps of obtaining and identifying for aplurality of test compounds.
 23. The method of claim 22, wherein saidsteps of obtaining, identifying, and repeating are performed in ahigh-throughput screening format.
 24. The method of claim 22, whereinsaid plurality of test compounds are taken from a chemical library. 25.The method of claim 22, further comprising: performing a quantitativestructure-activity relationship (QSAR) analysis on a set of selectedcompounds identified as potential ER-stress aggravating agent; using aresult of the QSAR analysis to design one or more new test compounds;and applying the obtaining and identifying steps on the new testcompound(s).
 26. A pharmaceutical composition useful for inducing oraggravating ER stress in a cell to trigger apoptosis in the cell,comprising: an ER-stress aggravating agent; and a pharmaceuticallyacceptable carrier.
 27. The pharmaceutical composition of claim 26,wherein said ER-stress aggravating agent is one selected from thecompounds according to claim
 5. 28. The pharmaceutical composition ofclaim 26, wherein said ER-stress aggravating agent is a prodrug capableof being metabolized into a SERCA inhibitor.
 29. The pharmaceuticalcomposition of claim 26, wherein said ER-stress aggravating agent iscapable of increasing the concentration of misfolded or damaged proteinsin the ER.
 30. The pharmaceutical composition of claim 29, wherein saidsecond ER-stress is a proteasome inhibitor or a protease inhibitor. 31.A pharmaceutical composition comprising a first ER-stress aggravatingagent according to the compounds of claim 5, and a second ER-stressaggravating agent capable of increasing the concentration of misfoldedor damaged proteins in the ER.
 32. A method of treating a diseasedcondition in a patient by inducing or aggravating ER stress in selecteddiseased cells to trigger apoptosis in the cells, comprising:administering a pharmaceutically effective amount of a pharmaceuticalcomposition according to claim
 26. 33. The method of claim 32, whereinsaid diseased cells are in a state of ER-stress prior to theadministrating step.
 34. The method of claim 32, further comprisingadministering a pharmaceutically effective amount of an apoptosisenhancer according to claim
 18. 35. The method of claim 32, furthercomprising administering a pharmaceutically effective amount of a secondpharmaceutical composition containing a different ER-stress aggravatingagent capable of increasing the concentration of misfolded or damagedproteins in the ER.
 36. The method of claim 35, wherein said differentER-stress aggravating agent is a proteasome inhibitor or a proteaseinhibitor.
 37. The method of claim 32, wherein said diseased conditionis caused by apoptosis disregulation.
 38. The method of claim 37,wherein said condition is cancer.
 39. The method of claim 38, whereinsaid cancer is glioblastoma multiforme.
 40. The method of claim 38,wherein said cancer is selected from breast carcinoma, pancreaticcarcinoma, Burkett's lymphoma, multiple myeloma, neuroblastoma, prostatecancer, colorectal cancer, recurring cancer, metastatic breast cancer,chemo-resistant tumors and drug-resistant cancer.
 41. A method ofaggravating stress in a cell's endoplasmic reticulum (ER) to triggerapoptosis, provided that the cell is in a state of low ER stress asevidenced by levels of GRP78 greater than the usual levels in normalcells, the method comprising: providing a compound that inhibits SERCAactivity in said cell resulting in the elevation of CHOP expressionwherein, the combination of inhibiting SERCA activity and elevating CHOPexpression results in a condition favorable for initiation of apoptosisin the cell.
 42. The method of claim 41, wherein said compound is not aCOX-2 inhibitor or a histamine-release agent.
 43. The method of claim41, where said compound is selected according to claim 5.